Generation of hemoglobin-based oxygen carriers using elastin-like polypeptides

ABSTRACT

Described herein is the use of elastin-like polypeptides to generate hemoglobin-based oxygen carriers as a means of preventing and treating conditions caused by blood loss or anemia, for example, hemorrhagic shock. Elastin-like polypeptides are capable of creating therapeutically functional fusion proteins through genetic engineering with a therapeutic agent, for example, hemoglobin and biologic equivalent thereof. Specific forms of these fusion proteins have the ability to form into spherical nanoparticles possessing a therapeutically agent at their core. This provides a unique basis for employing elastin-like polypeptides as hemoglobin carriers in the manufacture of blood substitutes.

CROSS REFERENCE TO RELATED APPLICATIONS

This application claims the benefit of priority to U.S. ProvisionalApplication No. 62/089,885, filed Dec. 10, 2014, entitled “GENERATION OFHEMOGLOBIN-BASED OXYGEN CARRIERS USING ELASTIN-LIKE POLYPEPTIDES”, thecontent of which is incorporated by reference herein in its entirety.

FIELD OF INVENTION

The present invention relates generally to artificial or synthetic bloodsubstitutes.

BACKGROUND OF THE INVENTION

The Circulatory System and the Nature of Hemoglobin

Blood, the means for delivering oxygen (O₂) and nutrients to the tissuesand removing carbon dioxide (CO₂) and waste products from the tissuesfor excretion, is composed of plasma in which red blood cells (RBCs orerythrocytes), white blood cells (WBCs), and platelets are suspended.The functions of blood can be grouped generally as maintenance ofintravascular volume, delivery of oxygen to tissues, provision ofcoagulation factors, provision of some defense mechanisms, andtransportation of metabolic waste products.

When the heart contracts, blood is pumped into certain major bloodvessels, and from there, continues through the circulatory system.Humans and other mammals have two-circuit circulatory systems: onecircuit is for pulmonary circulation (circulation to the lungs), and theother circuit is for systemic circulation (the rest of the body). Bloodthat is lacking oxygen is said to be deoxygenated. Deoxygenated blood,which has just exchanged oxygen for carbon dioxide across cellmembranes, and now contains mostly carbon dioxide, enters the rightatrium, where pulmonary circulation begins, and flows into the rightventricle. As the right ventricle contracts, it forces the deoxygenatedblood into the pulmonary artery, which carries the deoxygenated blood tothe lungs, where it becomes oxygenated.

Freshly oxygenated blood returns to the heart via the pulmonary veins,into the left atrium, which is where systemic circulation begins. Thefreshly oxygenated blood flows from the left atrium into the leftventricle. As the left ventricle contracts, the oxygenated blood ispumped into the main artery of the body—(the aorta), which branches intoother arteries, which then branch into smaller arterioles. Thearterioles meet up with capillaries, which bridge the smallest of thearteries (arterioles) and the smallest of the veins (venules). Near thearterial end, the capillaries allow materials essential for maintainingthe health of cells to diffuse out (water, glucose, oxygen, and aminoacids) and transport wastes and carbon dioxide to places in the bodythat can dispose of them. The waste products enter near the venous endof the capillary. Water diffuses in and out of capillaries to maintainblood volume, which adjusts to achieve homeostasis. Thereafter, thedeoxygenated blood travels through the venules and veins in its returnto the right atrium of the heart, which is where pulmonary circulationbegins.

Red blood cells comprise approximately 99% of the cells in blood, andtheir principal function is the transport of oxygen to and the removalof carbon dioxide from the tissues. About 95% of the dry weight of thered cell is hemoglobin. Hemoglobin functions primarily as a carrier of alarge volume of oxygen taken up in the lungs and delivered to thetissues.

The reversible oxygenation function of RBCs (i.e. a large volume ofoxygen taken up in the lungs and delivered to the tissues and theremoval of carbon dioxide) is carried out by hemoglobin. Hemoglobin iscomposed of about 6% heme and 94% globin (protein).

Human adult hemoglobin is a tetrameric protein comprising two alpha (α₁,α₂) and two beta (β₁, β₂) polypeptide subunits, each of which consistsof a polypeptide chain, globin, and an associated heme molecule. Heme isthe name given to the molecule of iron and the particular porphyrinfound in hemoglobin, for example, protoporphyrin IX. The alpha subunitconsists of 141 amino acids. The iron atom of the heme(ferroprotoporphyrin IX) group is bound covalently to the imidazole ofHis 87 (the “proximal histidine”) of the alpha subunit. The beta subunitis 146 residues long, and the heme group is bound to this subunit at His92. Hemoglobin forms a loose complex with oxygen when the iron is in theferrous (Fe⁺⁺) state. The four polypeptide subunits (α₁, α₂, β₁, β₂) areheld together by noncovalent attractions, for example, salt bridge,hydrogen bonds, and hydrophobic effect.

The primary amino acid structure of the human adult hemoglobin alpha andbeta subunits, and the nucleic acid sequences, which encode them, areknown (see Wilson et al., J. Biol. Chem., 1980, 255(7), 2807-2815)

The transport of oxygen from the body's external environment to itsperipheral tissues depends on several factors, including theconcentration and partial pressure of oxygen in the inspired air,alveolar ventilation, ventilation-perfusion relationships, cardiacoutput, blood volume, and hemoglobin concentration. FIG. 1 shows anoxygen dissociation curve, which is a plot of the proportion ofhemoglobin in its saturated form on the vertical axis against theprevailing oxygen tension on the horizontal axis. The position of theoxygen-hemoglobin dissociation curve describes the affinity ofhemoglobin for oxygen, and influences the transfer of oxygen fromhemoglobin in blood to tissue cells.

The oxygenated hemoglobin dissociation curve as shown in FIG. 1 has acharacteristic sigmoid shape, which is typical of allosteric proteinsdue to the cooperative effect that exists between the multiple oxygenbinding sites on the same hemoglobin molecule. When oxygen binds to thefirst subunit of deoxyhemoglobin, the first oxygen molecule increasesthe affinity of the remaining subunits for additional oxygen molecules.As additional oxygen is bound to the other hemoglobin subunits, oxygenbinding is incrementally strengthened, so that hemoglobin is fullyoxygen-saturated at the oxygen tension of lung alveoli. Likewise, oxygenis incrementally unloaded and the affinity of hemoglobin for oxygen isreduced as oxyhemoglobin circulates to deoxygenated tissue.

The value of percent (%) saturation can range from 0 (all sites empty)to 100% (all sites filled). Oxygen affinity can be characterized by aquantity P50, which is normal human adult partial pressure of oxygen atwhich 50% of sites are filled or at which 50% of the hemoglobin isoxygenated. For hemoglobin, P50 is 26 torrs. The oxygen dissociationcurve reflects the interaction between oxygen and hemoglobin, and boththe shape and position of the curve are subject to change by factorsthat modify the ability of hemoglobin to bind oxygen, including bodytemperature, pH of blood, CO₂ tension, and the concentration of2,3-diphosphoglycerate (2,3-DPG). Alterations in hemoglobin-oxygenaffinity also occur in many disease states. Table 1 summarizes factorsthat alter hemoglobin-oxygen affinity.

TABLE 1 Factors that increase or decrease P50. Increase P50 Decrease P50By Direct Effect: By Direct Effect: Increased [H+] Decreased [H+]Temperature Temperature PCO₂ PCO₂ DPG, ATP DPG, ATP Hgb Conc. Hgb Conc.Ionic Strength Ionic Strength Abnormal Hemoglobin Abnormal HemoglobinAldosterone Carboxy hemoglobin Methemoglobin

(Adapted from: Shappell, S. D. et al.: Adaptive, Genetic and IatrogenicAlterations of the Oxyhemoglobin dissociation Curve. Anesthesiology, 37:127-139, 1971)

The affinity of hemoglobin for oxygen depends on pH. The CO₂ moleculealso affects the oxygen-binding characteristics of hemoglobin. Both H+and CO₂ promote the release of bound O₂. Reciprocally, O₂ promotes therelease of bound H+ and CO₂.

The affinity of hemoglobin for oxygen is further regulated by organicphosphates, such as 2,3-bisphosphoglycerate (BPG). This highly anionicorganic phosphate is present in human red cells at about the same molarconcentration as hemoglobin. In the absence of BPG, the P50 ofhemoglobin is 1 torr. In its presence, P50 becomes 26 torrs. BPG lowersthe oxygen affinity of hemoglobin by a factor of 26, which is essentialin enabling hemoglobin to unload oxygen in tissue capillaries, bybinding to and cross-linking deoxyhemoglobin but not to the oxygenatedform. (Stryer, L, Portrait of an Allosteric Protein, Biochemistry, 4thed.) Certain diseases or age also affects affinity of hemoglobin foroxygen.

When hemoglobin's affinity for oxygen is increased, the RBCs havesubnormal P50 values and their oxyhemoglobin dissociation curves aresituated to the left of normal. These changes indicate that a lower thannormal oxygen tension will be needed to saturate the RBC hemoglobin inthe lung, and the release of oxygen in the tissue occurs at lower thannormal capillary oxygen tension. When hemoglobin's affinity for oxygenis decreased, the P50 values are higher and the oxyhemoglobindissociation curves are situated to the right of normal. These changesindicate that a higher than normal oxygen tension will be needed tosaturate hemoglobin in the lung, and that the release of oxygen in thetissue occurs at higher than normal capillary oxygen tension.

Within limits, rightward or leftward shifts of the oxygen dissociationcurves have little effect on arterial oxygen saturation since normalhuman adult arterial PO₂ is above 80 mm Hg. At the peripheral capillarylevel, however, even small shifts of the oxygen dissociation curve canbe important. A rightward shift of the curve indicates decreasedhemoglobin affinity for oxygen, while a leftward shift indicates anincrease in hemoglobin-oxygen affinity. A rightward shift of the curveis advantageous theoretically, since an equivalent amount of oxygen isreleased at a higher PO₂ than with a leftward positioned curve.

Blood Transfusions

Over 4.5 million patients require blood transfusions throughout NorthAmerica each year. Blood transfusions are a life-saving intervention fora number of clinical conditions including, without limitation, replacingblood lost during surgical procedures and following acute hemorrhage,for resuscitation procedures following traumatic injury, or for anemicpatients. In events involving acute trauma, occurring in a serious caraccident, for instance, a victim may need almost 100 pints of transfusedblood. Transfusion therapy has been an integral part of militarymedicine. As the most needed and vital component of blood, red bloodcells (RBCs) are the most transfused blood product in battlefield traumacare and more than 54,000 units of RBCs are transfused every year inmilitary hospitals. The primary goal of blood transfusion is to restorethe circulation of oxygen through the body, a function that isphysiologically mediated by the hemoglobin found in red blood cells. Itis reported that over 40% of all trauma-related deaths within the first24 hours results from hemorrhagic shock, which can be rapidly fatal;serious car accidents, battlefield injuries, and complications duringchild delivery are other examples of incidents leading to hemorrhagicshock. The overwhelming cause of mortality in each of these cases is aloss of oxygen-carrying blood. In such cases, the transportation timefrom the site of injury to a healthcare facility represents a criticaltime for the patient. However, blood transfusion is not readily donebefore reaching a hospital facility due to disadvantages and constraintsof blood transfusion that are discussed below.

Transfusion of a patient with donated blood, while used widely, has anumber of disadvantages. First, due to the irregular nature of blooddonations, blood supply shortages are common. Second, there may be ashortage of a patient's blood type. Third, transfused blood may becontaminated with infectious agents. Fourth, donated blood has a shortstored shelf life (42 days) and must be stored in a refrigeratedenvironment. Stored blood also loses 2,3-diphosphoglycerate (2,3-DPG) astime progresses, increasing its oxygen affinity and impairing oxygenunloading capacity in tissues. Fifth, complications can occur with bloodtransfusion due to inaccurate cross-matching, which remains the leadingdirect cause of death resulting from blood transfusion. Sixth, thegreatest risk of transfusion may be the alterations it induces inrecipients' immunological function. Multiple blood transfusions mayeventually lead to a severe systemic inflammatory response, which maycause increasing incidence of multiple organ failure.

Because of the many disadvantages and constraints of blood transfusionand shortages of blood supply, the need to develop a viable bloodsubstitute as an alternative to transfused blood has been longrecognized.

Blood Substitutes

Blood substitutes are the “Holy Grail” of trauma medicine thatresearchers have pursued for more than a century. The ideal bloodsubstitute would have none of the transfusion problems associated withblood, i.e., it would not require cross-matching or blood typing, couldbe stored preferably at room temperature for a long period, would have areasonable intravascular life span and thereafter be excreted promptly,and would be free of toxicity or disease transmission. It might be usedfor immediate restoration of oxygen delivery, such as in trauma, or inother urgent situations involving massive blood loss where red bloodcells are not available quickly. Since blood typing and cross-matchingwould not be necessary, the substitute might be carried in emergencyvehicles, stocked in emergency departments, or used by the military orcivilians in situations where access to blood is limited. Otherpotential uses of blood substitutes include organ perfusion andpreservation prior to transplantation, and improving oxygen delivery totissues that have an impaired blood supply. Unfortunately, to date, nooxygen-carrying blood substitutes are approved for use by the US Foodand Drug Administration (FDA).

Blood substitutes that have been developed previously can be groupedinto two categories: perfluorocarbon-based emulsions and cell-freehemoglobin-based blood substitutes. Perfluorochemical-based compositionsdissolve oxygen as opposed to binding it as a chelate as hemoglobindoes. They are chemically inert molecules containing, primarily,fluorine and carbon atoms and are capable of dissolving large amounts ofmany gases, including oxygen. However, most of the oxygen is releasedprior to reaching the oxygen-laden molecule in the capillary networkwhere the need for oxygen is greater. These molecules are hydrophobic innature, and hence have to be emulsified prior to intravenousadministration. Most of the development of these agents have been haltedor products been withdrawn from the market.

Products comprising modified cell-free hemoglobin, which are thought tobe more promising, are frequently referred to as hemoglobin-based oxygencarriers. Hemoglobin can be prepared in solution by lysis of red cells.The RBC membrane contains proteins, cholesterol and phospholipids.Stroma-free hemoglobin or acellular hemoglobin has been investigated asan oxygen carrier since the 1940s, when researchers realized that nativehemoglobin is not antigenic. A solution containing stroma-freehemoglobin has many advantages over intact red blood cells, includingthe ability to withstand sterilization and a shelf life of approximately2 years at room temperature for some products. However, stroma-freehemoglobin has many shortcomings. First, it is not as effective atoxygenation as are red blood cells, because free hemoglobin has reducedcontact with phosphates, causing the P50 curve to shift to the left,resulting in hemoglobin with a high oxygen affinity and limitedunloading. Second, when infused rapidly, stroma-free hemoglobin splitsinto dimers and is cleared by glomerular filtration and uptake by thereticuloendothelial system. Third, clinically, stroma-free hemoglobinhas been found to produce renal dysfunction, coagulopathy, andhypertension.

To address these limitations, a variety of approaches have been used tomolecularly stabilize and chemically modify hemoglobin. Bunncross-linked hemoglobin with bis (N-maleimidomethyl) ether (BME),reduced the hemoglobin molecule's tendency to form dimers, thusdecreasing its renal filtration and clearance, and prolonged itsintravascular retention (Bunn, J Exp Med. May 1, 1969; 129(5): 909-924).Other investigators have produced hemoglobin that had been chemicallymodified at the 2,3-DPG site, the amino terminal group, or internally inan attempt to prevent hemoglobin from disassociating into αβ dimers andas a means of restoring the P50 to near-normal levels. (Winslow R M,Hemoglobin modification. In: Winslow R M, editor. Blood Substitutes.London: Academic Press; 2006. pp. 341-53). Using a different approach,Bonsen et al. produced a hemoglobin that was polymerized withglutaraldehyde, which prolonged its intravascular retention (Bonsen P,Novel polymerized, cross-linked, stroma-free hemoglobin. United States:1975). Another modification approach involved the attachment ofhemoglobin to a larger molecule, which caused it to stay within thevascular system for a longer period of time than does non-modifiedhemogobin. In one study, hemoglobin coupled to dextran was shown tosupport life in dogs and cats in the absence of red blood cells. (Tam SC, Proc Natl Acad Sci USA. 1976 June; 73(6):2128-3114; Humphries R G, BrJ Pharmacol. 1980; 74:266).

Out of these and other suggested chemically modified hemoglobins,several products progressed to human studies and limited testing inhuman patients. However, only a few advanced to Phase II and III trials:DCLHb/HemAssist® (Baxter), SFH-P/PolyHeme® (Northfield), andHBOC-201/Hemopure® (Biopure). (Chen, J Y, et al., Clinics 2009,64(8):803-13); and Grethlein, 2012,(http://emedicine.medscape.com/article/207801-overview#a1).

Diaspirin Cross-Linked Hemoglobin (DCLHb/HemAssist)® consists ofhemoglobin with cross-linking between the two alpha chains, which lendsstability to the molecule. The source of hemoglobin consisting ofoutdated human red blood cells that were pooled, washed, lysed andfiltered. The product is then deoxygenated, crosslinked withbis(3,5-dibromosalicyl)fumarate (DBBF), and reoxygenated. DCLHbsolutions exhibits a P50 of 32 mmHg. It also exhibits a long shelf lifewhen stored in a freezer. However, Baxter Healthcare halted furtherdevelopment of DCLHb in 1998 after the product failed trials in patientswith stroke and trauma. (Winslow R M. Current status of oxygen carriers(‘blood substitutes’): 2006. Vox Sang. August 2006; 91 (2): 102-10.)

SFH-P/PolyHeme® (Northfield Laboratories Inc., Evanston, Ill.) isproduced by crosslinking stroma-free hemoglobin from outdated RBCs withglutaraldehyde and then pyridoxylating it. The product has a P50 of20-22 mmHg (compared to a normal RBC, which exhibits a P50 of 26 mmHg).In May 2009, the FDA refused to approve PolyHeme.

HBOC-201/Hemopure® is derived from bovine hemoglobin polymerized withglutaraldehyde. HBOC-201's P50 is 40 mmHg, resulting in a lower oxygenaffinity than native hemoglobin. It has an intravascular half-life of8-23 hours and a shelf life of 36 months at room temperature. Hemopure®is approved in South Africa for the treatment of adult surgical patientswho are acutely anemic with the intention of eliminating or reducing theneed for allogenic red blood cell transfusions. In the United States,phase II trials have been put on hold due to safety issues. Hemopure®was removed from the market in 2008 due to deaths related to kidneyfailure following transfusion of the product.

Other first-generation polymerized hemoglobin products include HemoLink®(Hemosol Corporation, Mississauga, Canada), a human hemoglobin basedoxygen carrier, containing polymerized human Hb, cross-linked witho-raffinose.

Hemospan® (Sangart Inc., San Diego, Calif.), also known as MP4OX, is anacellular PEG-conjugated human hemoglobin therapeutic in clinical trialsin Europe and the United States. The product is prepared bysite-specific conjugation of maleimide-activated poly(ethylene) glycol(PEG, MW ˜5500) to human oxyhemoglobin through maleimidation reactionseither (1) directly to reactive Cys thiols or (2) at surface Lys groupsfollowing thiolation using 2-iminothiolane. The thiolation/maleimidationreactions lead to the addition of ˜8 PEGs per hemoglobin tetramer. (KimD. Vandegriff, Bioconjugate Chem., 2008, 19 (11), pp 2163-2170) Inanimal models, Hemospan (MP4OX) has been shown to be effective in casesof hemorrhagic shock.

Pyridoxylated hemoglobin polyoxyethylene conjugate (PHP) is a conjugatedhemoglobin developed by Apex Bioscience that completed a phase III trialin August 2009 in patients with shock associated with systemicinflammatory response syndrome (SIRS) to evaluate the safety andefficacy of continuous IV infusion of PHP plus conventional vasopressortreatment versus continuous IV infusion of Plasma-lyte A plusconventional vasopressors as a treatment for restoring hemodynamicstability in patients.

A recombinant 130 kDa dihemoglobin, which is made up of a single-chaintetra-α globin and four β globins has been expressed as a solubleprotein in E. coli. (Marquardt, et al., J. Funct. Biomater. 2012, 3(1),61-78). A 260 kDa tetrahemoglobin has also been produced by chemicalcrosslinking of a dihemoglobin that contains a Lys16Cys mutation in theC-terminal α-globin subunit. Tetrahemoglobin also shows reducedvasoactivity in conscious rats that is comparable to that observed fordihemoglobin. (Marquardt, et al., J. Funct. Biomater. 2012, 3(1), 61-78)

Efforts have been made to encapsulate hemoglobin within a lipid-membrane(e.g. liposome) to create a compound capable of carrying oxygen whilenot being associated with significant vasoconstriction. However, thehalf-life of this liposome encapsulated hemoglobin is short, which hashindered its clinical development. Liposome encapsulated hemoglobin isprone to aggregate and fuse together after several days of storage,diminishing its functionality. At present, the only institutions workingactively on this product are in Japan.

Biodegradable polymers are often considered as alternatives to lipidsfor their improved in vivo stability. A vast array of biodegradablepolymers, ranging from synthetic to natural and to hybrid orrecombinant, have been studied and developed for drug delivery.Depending on the choice of building blocks, block polymers can assembleto nanostructures in the form of micelles, electrostatic complexes, orpolymersomes. (Hoffman A S, J. of controlled release: official journalof the controlled release society, 2008, 132:153-163).

Synthetic polymers include degradable or non-degradable syntheticpolymers. Exemplary synthetic degradable polymers includepoly(c-caprolactone) (PCL), poly(ε-caprolactone-co-ethyl ethylenephosphate) (PCLEEP), poly(lactic acid) (PLA), poly(lactic-co-glycolicacid) (PLGA), poly(lactic acid-co-ε-caprolactone) (PLACL), andpolydioxanone (PDO). Exemplary non-degradable polymers include polyacrylamide (PAAm), poly acrylic acid (PAA), poly acrylonitrile (PAN),poly amide (Nylon) (PA, PA-4,6, PA-6,6), poly aniline (PANI), polybenzimidazole (PBI), poly bis(2,2,2-trifluoroethoxy) phosphazene, polybutadiene (PB), poly carbonate (PC), poly ether amide (PEA), poly etherimide (PEI), poly ether sulfone (PES), poly ethylene (PE), polyethylene-co-vinyl acetate (PEVA), poly ethylene glycol (PEG), polyethylene oxide (PEO), poly ethylene terephthalate (PET), polyferrocenyldimethylsilane (PFDMS), poly 2-hydroxyethyl methacrylate(HEMA), poly 4-methyl-1-pentene (TpX), poly methyl methacrylate (pMMA),poly p-phenylene terephthalamide (PPTA), poly propylene (PP), polypyrrole (PPY), poly styrene (PS), polybisphenol-A sulfone (PSF), polysulfonated styrene (PSS), Styrene-butadiene-styrene triblock copolymer(SBS), poly urethane (PU), poly tetrafluoro ethylene (PTFE), poly vinylalcohol (PVA), poly vinyl carbazole, poly vinyl chloride (PVC), polyvinyl phenol (PVP), poly vinyl pyrrolidone (PVP), and poly vinylidenedifluoride (PVDF). A preferred synthetic polymer is polyethersulfone(PES).

Natural polymers are biocompatible and biodegradable and are derivedfrom biological systems including protein polymers, DNA, andpolysaccharides. They possess low toxicity and potentially favorablepharmacokinetics in the circulation.

Protein polymers can be synthetic or natural, or recombinant.

Recombinant protein polymers comprise repetitive amino acid sequencesthat can spontaneously self-assemble into sub-100 nm size nanoparticlesupon conjugation of diverse hydrophobic molecules. Recombinant proteinpolymers or genetically engineered protein polymers are biodegradableand potentially biocompatible if the artificial sequence is notantigenic. Genetic engineering allows precise control over structuraland functional properties of recombinant proteins, such as theirmolecular weight, solubility, hydrophobicity, targeting motif, secondarystructures, and drug conjugation sites. Two potential recombinantprotein systems are elastin-like polypeptides (ELPs), andsilk-elastin-like polypeptides (SELPs).

ELPs, a family of recombinant proteins derived from human tropoelastin,are one class of artificial repetitive polypeptides which have grown inpopularity as an alternative to synthetic polymers. The basic buildingblock is a short hydrophobic domain, comprised of a five amino acidmotif (Val-Pro-Gly-Xaa-Gly)n. By substituting the fourth amino acid Xaain the pentapeptide, ELPs can undergo reversible and rapid phasetransition in response to temperature. ELPs undergo an inverse phasetransition above a transition temperature (Tt), which is primarily afunction of the guest residue Xaa, n, and concentration (Urry D W.Journal of Physical Chemistry B. 1997, 101:11007-11028; Chilkoti A.Biomacromolecules. 2004, 5:846-851). In solution, ELPs are structurallydisordered. When the temperature is raised above their Tt, they undergoa sharp (2-3° C. range) phase transition, leading to biopolymercoacervation (Urry D W. Journal of Physical Chemistry B. 1997,101:11007-11028). This process is fully reversible when the temperatureis lowered below Tt. Phase separation can be triggered by other externalstimuli such as changes in ionic strength, pH, solvent, and magneticfields (Chilkoti A, Advanced Drug Delivery Reviews. 2002, 54:1093-1111;Mackay J A, Biomacromolecules. 2010, 11 (11):2873-2879).

A series of ELPs with distinct transition temperatures have beendesigned as drug carriers (MacKay J A, Nat Mater, 2009, 8:993-999). Forexample, in one system, ELP-peptide fusion protein was conjugated todoxorubicin (Dreher M R, Cancer Res. 2007, 67:4418-4424), which formedmicelles and aggregated in the tumor microenvironment under hyperthermicconditions leading to increased accumulation at the tumor site. Inanother study, the effect of hyperthermia-induced micelle formation wasexploited to present multivalent targeting motifs to enhance cellularuptake (Dreher M R, J. Am Chem Soc. 2008, 130:587-694). Multiblock ELPshave been developed for drug delivery in the form of nanoparticles (NP)or a hydrogel depending on the multiblock composition and processingmethod (Sallach R E, Biomaterials 2009, 30:409-422; Kim W, Adv DrugDeliv Rev. 2010, 62:1468-1478; Jordan S W, Biomaterials 2007,28:1191-1197; Wu X, Biomacromolecules 2008, 9:1787-1794).

Based on prior studies in small animal models, ELPs have lowimmunogenicity (Megeed Z, et al. Adv Drug Delivery Rev. 2002,54:1075-1091; Cappello J, et al. J Cont Rel. 1998; 53:105-117; Liu W, etal. J Control Release. 2006; 116:170-178).

Because ELPs can be produced via genetic engineering, their composition,MW, and polydispersity can be precisely controlled. ELPs can bereproduced with high yield (˜100-200 mg/L) in E. coli, and can berapidly purified by exploiting their phase transition behavior, so thathigh-purity, clinical grade material is obtained.

FIG. 2 is a transmission electron microscopy (TEM) of negatively stainedwith uranylacetate ELP micelle nanoparticles formed by repetitiveamino-acid sequences with different guest residues in hydrophobic andhydrophilic blocks (white round objects) with an average particlediameter of about 33 nm. (S. M. Janib et al., Integr Biol, 2013,5(1):183-194).

As a peptide therapeutic, ELP biopolymers have reasonably goodpharmacokinetics with terminal circulation half-lives of 8-11 h in nudemice (Liu W, J Control Release. 2006; 116:170-178). A 59 kD ELPnanoparticle [V5A2G3-150] with a transition temperature >37° C.evaluated in mice (as shown in FIG. 3), exhibited an eliminationhalf-life of 6-8 hrs in mouse serum (MacKay, J A, Int J Hyperthermia,2008, 24(6):483).

FIG. 4 shows uptake and degradation of ELP nanoparticles in transformedhepatocytes. This in vitro study demonstrated that mice hepatocytesenzymatically degrade ELP nanoparticles (M. Shah et al. Protein Sci,2012, 21(6): 743-750).

Drugs conjugated with ELPs gain properties of thermally-induced phasetransition and also maintain their in vitro bioactivity. This has beenshown for chemically-conjugated chemotherapeutics such as doxorubicin(Dreher M R, J Control Release. 2003; 91(1-2):31-43), recombinantoligopeptide fusions with cell penetrating peptides (Massodi I, JControl Release. 2005; 108(2-3):396-408), a c-myc oncogene inhibitor,(Bidwell G L, Mol Cancer Ther. 2005; 4(7): 1076-1085) and recombinantprotein fusions with interleukin-1 receptor antagonist (Shamji M F,Arthritis Rheum. 2007; 56(11):3650-3661) and other proteins(Trabbic-Carlson K, Protein Sci. 2004; 13(12):3274-3284; Trabbic-CarlsonK, Protein Eng Des Sel. 2004; 17(1):57-66). Surfaces coated with an ELPfused to the RGD or fibronectin CS5 cell binding sequence also retain anability to support in vitro endothelial cell adhesion and spreading.(Liu J C, Biomacromolecules. 2004; 5(2):497-504). Other applications ofELPs, including entrapment of small molecules such as dexamethasone,(Herrero-Vanrell R, J Control Release. 2005; 102(1):113-122) have alsobeen investigated. (Simnick A J, Polymer Reviews. 2007; 47:121-154).

ELPs are attractive as hemoglobin delivery systems for at least fiveimportant reasons: first, because ELPs can be genetically encoded, theirsynthesis from a synthetic gene in a heterologous host (e.g., bacteriaor eukaryotic cell) can provide complete control over the amino acidsequence and molecular weight, two variables that are not easy toprecisely control in synthetic polymers. Second, ELPs can be expressedfrom a plasmid-borne gene in E. coli to relatively high yields (˜500mg/L growth), which also makes them attractive for hemoglobin deliveryapplications where large quantities of polymer are often required.Third, they can be purified from E. coli—and other—cell lysates in batchprocess by exploiting their inverse temperature phase transition withoutthe need for chromatography, which simplifies large scale purificationof ELPs (Meyer D E, et al., Nat Biotechnol. 1999, 17:1112-1115). Fourth,ELPs can be engineered to approach the viscoelastic properties of nativeelastin upon crosslinking. Fifth, they are biocompatible, biodegradable,and non-immunogenic (Urry D W, et al., J Bioact Compat Polym. 1991,6:263-282).

Silk proteins are produced by a variety of insects and spiders, and formfibrous materials in nature, such as spider orb webs and silkwormcocoons. Silk protein is a native block copolymer with alternating largehydrophobic and hydrophilic blocks. The hydrophobic block is generally arepetitive sequence conserved with short-chain amino acids, such asglycine and alanine. The hydrophilic block is less conserved and usuallycontains non-repetitive sequences rich in charged amino acids. Thehydrophilic domain is often substituted with other peptide sequences toachieve specific function for drug delivery. The length of thehydrophobic domain can also be tuned to yield protein NPs withreproducible sizes for drug and gene delivery (Numata K, Biomaterials2007, 28:1191-1197; Numata K, Adv Drug Deliv Rev. 2010, 62:1497-1508). Arecent study demonstrated that a SELP recombinant protein endowed with acell penetrating peptide could achieve transfection efficiency 45 timeshigher than that of poly(ethyleneimine). (Numata K, Silk-based GeneCarriers with Cell Membrance Destabilizing Peptides, Biomacromolecules2010).

Silkworm silk from B. mori silkworm silk-like repeats of GAGAGS andelastin block (VPGVG) copolymers, and silk-elastin-like proteins (SELP)constructed by recombinant DNA techniques, have been utilized as geneand drug delivery systems, by forming hydrogels to release adenoviruscontaining reporter genes.

Many clinical trials involving blood substitutes have been discontinuedor held because they induced adverse effects including vasoconstriction,hypertension, or liver failure due to metabolic byproducts. Therefore,new strategies to discover or biosynthesize biocompatible materials,which can deliver oxygen with improved therapeutic efficacy andnontoxicity are needed.

SUMMARY OF THE INVENTION

According to one aspect, the described invention provides abiocompatible pharmaceutical composition comprising a therapeutic amountof a complex comprising a polymer in association with a hemoglobin (Hb),a Hb subunit(s), a Hb fragment(s), a Hb derivative(s), or a functionalequivalent thereof that stores and releases oxygen in accordance with anoxygen dissociation curve; wherein the therapeutic amount of the complexis effective to treat a condition caused by blood loss, anemia, or ahemoglobin disorder, and to improve subject survival relative to acontrol, wherein the polymer is a protein polymer, a polynucleotidepolymer, a polysaccharide polymer, or a synthetic polymer.

According to one embodiment, the condition caused by blood loss includeshemorrhagic shock.

According to one embodiment, the protein polymer is associated with theHb, the Hb subunit(s), the Hb fragment(s), the Hb derivative(s), or thefunctional equivalent thereof via a covalent bond, an ionic bond, ahydrogen bond, a hydrophobic force, encapsulation, or via fusion.According to another embodiment, the protein polymer is an elastin-likepolypeptide (ELP).

According to one embodiment, the ELP and the Hb, the Hb subunit(s), theHb fragment(s), the Hb derivative(s), or the functional equivalentthereof are operatively linked to form a fusion protein, which isencoded by a polynucleotide comprising a nucleotide sequence thatencodes the ELP and a nucleotide sequence that encodes the Hb, the Hbsubunit(s), the Hb fragment(s), the Hb derivative(s), or the functionalequivalent thereof. According to another embodiment, the ELP and the Hb,the Hb subunit(s), the Hb fragment(s), the Hb derivative(s), or thefunctional equivalent thereof are operatively linked to form a fusionprotein, which is obtained by chemically joining the ELP and the ELP andthe Hb, the Hb subunit(s), the Hb fragment(s), the Hb derivative(s), orthe functional equivalent thereof. According to another embodiment, theELP and the Hb, the Hb subunit(s), the Hb fragment(s), the Hbderivative(s), or the functional equivalent thereof are operativelylinked to form a complex, wherein the ELP is assembled into a sphericalnanoparticle comprising a core into which the Hb, the Hb subunit(s), theHb fragment(s), the Hb derivative(s), or the functional equivalentthereof is encapsulated.

According to one embodiment, the fusion protein is assembled into aspherical nanoparticle comprising a core inside of which the Hb, the Hbsubunit(s), the Hb fragment(s), the Hb derivative(s), or the functionalequivalent thereof is enclosed.

According to one embodiment, the ELP comprises a pentameric amino acidmotif (Val-Pro-Gly-Xaa-Gly)n, wherein Xaa specifies any amino acid and ndenotes a number of repetitive motifs. According to another embodiment,n=20-90, and Xaa is Serine or a conservative amino acid substitutethereof. According to another embodiment, the conservative amino acidsubstitute of Serine is Thr. According to another embodiment, n=20-90,and Xaa is Isoleucine or a conservative amino acid substitute thereof.According to another embodiment, the conservative amino acid substituteof Isoleucine is Leu or Met or Val.

According to one embodiment, the ELP comprises a diblock copolymercomprising: a hydrophilic block comprising a pentameric amino acid motif(Val-Pro-Gly-Xaa-Gly)n, wherein n=20-90, and Xaa is a hydrophilic aminoacid; and a hydrophobic block comprising a pentameric amino acid motif(Val-Pro-Gly-Xaa-Gly)n, wherein n=20-90, and Xaa is a hydrophobic aminoacid. According to another embodiment, for the hydrophilic block, theXaa is selected from the group consisting of lysine (+), arginine (+),aspartate (−) and glutamate (−), serine, threonine, asparagine,glutamine, and histidine; and for the hydrophobic block, Xaa is selectedfrom the group consisting of alanine, valine, leucine, isoleucine,proline, phenylalanine, tryptophan, and methionine. According to anotherembodiment, for the hydrophilic block the Xaa is Serine or aconservative amino acid substitute thereof; and for the hydrophobicblock the Xaa is Isoleucine or a conservative amino acid substitutethereof. According to another embodiment, the conservative amino acidsubstitute of Serine is Thr; and the conservative amino acid substituteof Isoleucine is Leu or Met or Val. According to another embodiment,n=48 for hydrophobic block and n=48 for hydrophilic block.

According to one embodiment, the Hb, the Hb subunit(s), the Hbfragment(s), the Hb derivative(s), or the functional equivalent thereofis operatively linked to the C-terminus of the ELP. According of anotherembodiment, the Hb, the Hb subunit(s), the Hb fragment(s), the Hbderivative(s), or the functional equivalent thereof is operativelylinked to the hydrophobic block of the ELP. According to anotherembodiment, the Hb, the Hb subunit(s), the Hb fragment(s), the Hbderivative(s), or the functional equivalent thereof is of an amino acidsequence selected from the group consisting of SEQ ID No. 4, SEQ ID No.5 and SEQ ID No. 6. According to another embodiment, the ELP is of aminoacid sequence SEQ ID NO. 7. According to another embodiment, the Hb, theHb subunit(s), the Hb fragment(s), the Hb derivative(s), or thefunctional equivalent thereof is encoded by a polynucleotide sequenceselected from the group consisting of SEQ ID No. 1, SEQ ID No. 2 and SEQID No. 3.

According to one embodiment, the biocompatible pharmaceuticalcomposition further comprises one or more pharmaceutically acceptablesalts.

According to another aspect, the describe invention provides a method oftreating a condition due to blood loss and improving subject survival,the method comprising: (1) administering a biocompatible pharmaceuticalcomposition comprising a therapeutic amount of a complex comprising apolymer associated with a Hb, subunit(s), a Hb fragment(s), a Hbderivative(s), or a functional equivalent thereof, wherein the polymeris a protein polymer, a polynucleotide polymer, a polysaccharidepolymer, or a synthetic polymer; wherein the therapeutic amount iseffective to store and release oxygen in accordance with an oxygendissociation curve.

According to one embodiment, the condition caused by blood loss includeshemorrhagic shock.

According to one embodiment, the protein polymer is associated with theHb, the Hb subunit(s), the Hb fragment(s), the Hb derivative(s), or thefunctional equivalent thereof via a covalent bond, an ionic bond, ahydrogen bond, a hydrophobic force, encapsulation, or via fusion.According to another embodiment, the protein polymer is an elastin-likepolypeptide (ELP). According to another embodiment, the ELP and the Hb,the Hb subunit(s), the Hb fragment(s), the Hb derivative(s), or thefunctional equivalent thereof are operatively linked to form a fusionprotein, which is encoded by a polynucleotide comprising a nucleotidesequence that encodes the ELP and a nucleotide sequence that encodes theHb, the Hb subunit(s), the Hb fragment(s), the Hb derivative(s), or thefunctional equivalent thereof. According to another embodiment, the ELPand the Hb, the Hb subunit(s), the Hb fragment(s), the Hb derivative(s),or the functional equivalent thereof are operatively linked to form afusion protein, which is obtained by chemically joining the ELP and theELP and the Hb, the Hb subunit(s), the Hb fragment(s), the Hbderivative(s), or the functional equivalent thereof. According toanother embodiment, the ELP is assembled into a spherical nanoparticlecomprising a core inside of which the Hb, the Hb subunit(s), the Hbfragment(s), the Hb derivative(s), or the functional equivalent thereofis encapsulated.

According to one embodiment, the fusion protein is assembled into aspherical nanoparticle comprising a core inside of which the Hb, the Hbsubunit(s), the Hb fragment(s), the Hb derivative(s), or the functionalequivalent thereof is enclosed.

According to one embodiment, the ELP comprises a pentameric amino acidmotif (Val-Pro-Gly-Xaa-Gly)n, wherein Xaa specifies any amino acid and ndenotes a number of repetitive motifs. According to another embodiment,n=20-90, and Xaa is Serine or a conservative amino acid substitutethereof. According to another embodiment, the conservative amino acidsubstitute of Serine is Thr. According to another embodiment, whereinn=20-90, and Xaa is Isoleucine or a conservative amino acid substitutethereof. According to another embodiment, the conservative amino acidsubstitute of Isoleucine is Leu or Met or Val.

According to one embodiment, the ELP comprises a diblock copolymercomprising: a hydrophilic block comprising a pentameric amino acid motif(Val-Pro-Gly-Xaa-Gly)n, wherein n=20-80, and Xaa is a hydrophilic aminoacid; and a hydrophobic block comprising a pentameric amino acid motif(Val-Pro-Gly-Xaa-Gly)n, wherein n=20-80, and Xaa is a hydrophobic aminoacid. According to another embodiment, the Xaa is selected from thegroup consisting of lysine (+), arginine (+), aspartate (−) andglutamate (−), serine, threonine, asparagine, glutamine, and histidinein the hydrophilic block; and Xaa is selected from the group consistingof alanine, valine, leucine, isoleucine, proline, phenylalanine,tryptophan, and methionine in the hydrophobic block. According toanother embodiment, the hydrophilic block the Xaa is Serine or aconservative amino acid substitute thereof; and for the hydrophobicblock the Xaa is Isoleucine or a conservative amino acid substitutethereof. According to another embodiment, the conservative amino acidsubstitute of Serine is Thr, and the conservative amino acid substituteof Isoleucine is Leu or Met or Val. According to another embodiment,n=48 for hydrophobic block and n=48 for hydrophilic block.

According to one embodiment, the Hb, the Hb subunit(s), the Hbfragment(s), the Hb derivative(s), or the functional equivalent thereofis operatively linked to the C-terminus of the ELP. According to anotherembodiment, the Hb, the Hb subunit(s), the Hb fragment(s), the Hbderivative(s), or the functional equivalent thereof is operativelylinked to the hydrophobic block of the ELP. According to anotherembodiment, the Hb, the Hb subunit(s), the Hb fragment(s), the Hbderivative(s), or the functional equivalent thereof is of amino acidsequence selected from the group consisting of SEQ ID No. 4, SEQ ID No.5 and SEQ ID No. 6. According to another embodiment, the ELP is of aminoacid sequence SEQ ID NO. 7. According to another embodiment, the Hb, theHb subunit(s), the Hb fragment(s), the Hb derivative(s), or thefunctional equivalent thereof is encoded by a polynucleotide sequenceselected from the group consisting of SEQ ID No. 1, SEQ ID No. 2 and SEQID No. 3.

According to one embodiment, the biocompatible pharmaceuticalcomposition further comprises one or more pharmaceutically acceptablesalts.

According to one embodiment, the method further comprises constructing avector and/or host cell comprising a fusion gene polynucleotide thatcomprises a polynucleotide sequence coding a fusion protein comprisingELP and Hb, the Hb subunit(s), the Hb fragment(s), the Hb derivative(s),or the functional equivalent thereof.

According to one embodiment, the method further comprises preparing thefusion protein by expressing the fusion gene polynucleotide in anexpression system.

According to one embodiment, the method further comprises separating orpurifying the fusion protein from the expression system.

According to one embodiment, the method further comprises preparing thefusion protein by chemically operatively linking the ELP and Hb, the Hbsubunit(s), the Hb fragment(s), the Hb derivative(s), or the functionalequivalent thereof.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 The oxyhemoglobin dissociation curve plots the proportion ofhemoglobin in its saturated form on the vertical axis against theprevailing oxygen tension on the horizontal axis.

FIG. 2 is a transmission electron microscopy (TEM) of negatively stainednanoparticles (white round objects) with an average particle diameter ofabout 33 nm stained with uranyl acetate. (S. M. Janib et al., IntegrBiol, 2013)

FIG. 3 shows that polypeptide nanoparticles exhibit an eliminationhalf-life of 6-8 hrs in mouse serum (J. A. MacKay and A. Hilkoti, Int JHyperthermia, 2008)

FIG. 4 shows in vitro studies demonstrating that mice hepatocytesenzymatically degrade nanoparticles (M. Shah et al. Protein Sci, 2012)

FIG. 5 is a schematic of the chemical conjugation process used to linkelastin-like polypeptides (ELPs) to hemoglobin.

FIG. 6 shows SDS-PAGE of ELP-hemoglobin fusions. M: molecular weightladder; Lanes 1-2: ELP-hemoglobim fusion (2:1 ratio of hemoglobin:ELP);Lanes 3-4: ELP-hemoglobin fusion (1:1 ratio of hemoglobin:ELP); Lanes5-6: ELP-hemoglobin fusion (1:4 ratio of hemoglobin:ELP); Lanes 7-8:hemoglobin; Lanes 9-10: ELP.

FIG. 7 shows a chromatogram of a size exclusion analysis of anELP-hemoglobin fusion (1:4 ratio of hemoglobin:ELP). The first peak(Fraction 1) is ELP-hemoglobin fusion. The second peak (Fraction 2) isELP.

FIG. 8 shows a bar graph (intensity (%) vs. radium (nm)) of dynamiclight scattering (DLS) results for Fraction 1 (first peak) and Fraction2 (second peak) of the size exclusion analysis shown in FIG. 7.Hydrodynamic radius of Fraction 1=11.4 nm. Hydrodynamic radius ofFraction 2=7.4 nm.

FIG. 9 shows a line graph (intensity (%) vs. radium (nm)) of dynamiclight scattering (DLS) results for hemoglobin, ELP and ELP-hemoglobinfusion.

FIG. 10 shows UV-vis results (absorbance vs. wavelength in nm) forFraction 1 (first peak) and Fraction 2 (second peak) of the sizeexclusion analysis shown in FIG. 7. The UV-vis results indicate that 400nm absorption of hemoglobin was maintained after ELP modification.

FIG. 11 shows phase separation results (absorbance at 350 nm vs.Temperature in ° C.) for Fraction 1 (first peak) and Fraction 2 (secondpeak) of the size exclusion analysis shown in FIG. 7. The phaseseparation results indicate that ELP phase separation is maintainedafter ELP-hemoglobin fusion.

DETAILED DESCRIPTION OF THE INVENTION Definitions

The terms “administering” or “administration” as used herein are usedinterchangeably to mean the giving or applying of a substance andinclude in vivo administration, as well as administration directly totissue ex vivo.

The terms “amino acid residue” or “amino acid” or “residue” are usedinterchangeably to refer to an amino acid that is incorporated into aprotein, a polypeptide, or a peptide, including, but not limited to, anaturally occurring amino acid and known analogs of natural amino acidsthat can function in a similar manner as naturally occurring aminoacids.

The abbreviations used herein for amino acids are those abbreviations,which are conventionally used: A=Ala=Alanine; R=Arg=Arginine;N=Asn=Asparagine; D=Asp=Aspartic acid; C=Cys=Cysteine; Q=Gln=Glutamine;E=Glu=Glutamic acid; G=Gly=Glycine; H=His=Histidine; I=Ile=lsoleucine;L=Leu=Leucine; K=Lys=Lysine; M=Met=Methionine; F=Phe=Phenyalanine;P=Pro=Proline; S=Ser=Serine; T=Thr=Threonine; W=Trp=Tryptophan;Y=Tyr=Tyrosine; V=Val=Valine. The amino acids may be L- or D-aminoacids. An amino acid may be replaced by a synthetic amino acid, which isaltered so as to increase the half-life of the peptide or to increasethe potency of the peptide, or to increase the bioavailability of thepeptide.

Based on its propensity to be in contact with polar solvent like water,a side chain may be classified as hydrophobic (low propensity to be incontact with water), polar or charged (energetically favorable contactwith water or hydrophilic). The charged amino acid residues includelysine (+), arginine (+), aspartate (−) and glutamate (−). Polar aminoacids include serine, threonine, asparagine, glutamine, and histidine.The hydrophobic amino acids include alanine, valine, leucine,isoleucine, proline, phenylalanine, tryptophan, and methionine. Cysteinemay be considered slightly polar or nonpolar. Tyrosine may be consideredpolar (due to hydroxyl group on phenyl ring in side chain) or non-polar(due to aromatic ring).

The following represent groups of amino acids that are conservativesubstitutions for one another:

Alanine (A), Serine (S), Threonine (T);

Aspartic Acid (D), Glutamic Acid (E);

Asparagine (N), Glutamine (Q);

Arginine (R), Lysine (K);

Isoleucine (I), Leucine (L), Methionine (M), Valine (V); and

Phenylalanine (F), Tyrosine (Y), Tryptophan (W).

The term “amphiphilic” as used herein refers to a compound containing alarge organic cation or anion, which possesses a long unbranchedhydrocarbon chain, e.g. CH₃(CH₂)nCO₂-M+, CH₃(CH₂)nN+(CH₃)₃X— (n>7),CH₃(CH₂)nSO₃-M+. The existence of distinct polar (hydrophilic) andnonpolar (hydrophobic) regions in the molecule promotes the formation ofmicelles in dilute aqueous solution.

The term “associate” and its various grammatical forms as used hereinrefers to joining, connecting, or combining to, either directly,indirectly, actively, inactively, inertly, non-inertly, completely orincompletely.

The term “biocompatible” as used herein refers to causing no clinicallyrelevant tissue irritation, injury, toxic reaction, or immunologicalreaction to living tissue.

The term “biodegradable” as used herein refers to material that willbreak down actively or passively over time by simple chemical processes,by action of body enzymes or by other similar biological activitymechanisms.

The term “block” as used herein refers to a portion of a macromolecule,comprising many constitutional units, that has at least one feature,which is not present in the adjacent portions.

The term “block copolymer” as used herein refers to a copolymer that isa block polymer. In a block copolymer, adjacent blocks areconstitutionally different, i.e., each of these blocks comprisesconstitutional units derived from different characteristic species ofmonomer or with different composition or sequence distribution ofconstitutional units.

The term “blood substitutes” as used herein refers to an oxygen storageand delivery therapeutic. One type of the artificial blood substitute isa “hemoglobin-based oxygen carrier”.

The term “carrier” as used herein refers to a usually inactive substancethat acts as a vehicle for an active substance. The terms “excipient”,“vehicle”, or “carrier” refer to substances that facilitate the use of,but do not deleteriously react with, the active compound(s) when mixedwith it. The term “active” refers to the ingredient, component orconstituent of the compositions of the present invention responsible forthe intended therapeutic effect. Carriers must be of sufficiently highpurity and of sufficiently low toxicity to render them suitable foradministration to the subject being treated. The carrier can be inert,or it can possess pharmaceutical benefits. The term “pharmaceuticallyacceptable carrier” as used herein refers to any substantially nontoxiccarrier conventionally useful for administration of pharmaceuticals inwhich the active component will remain stable and bioavailable. Thepharmaceutical compositions within the described invention contain atherapeutically effective amount of included in apharmaceutically-acceptable carrier. The term“pharmaceutically-acceptable carrier” as used herein refers to one ormore compatible solid or liquid filler, diluents or encapsulatingsubstances which are suitable for administration to a human or othervertebrate animal. The term “carrier” as used herein refers to anorganic or inorganic ingredient, natural or synthetic, with which theactive ingredient is combined to facilitate the application. Thecomponents of the pharmaceutical compositions also are capable of beingcommingled in a manner such that there is no interaction which wouldsubstantially impair the desired pharmaceutical efficiency.

The term “cell” is used herein to refer to the structural and functionalunit of living organisms and is the smallest unit of an organismclassified as living.

The term “cell culture” as used herein refers to establishment andmaintenance of cell populations in vitro derived from dispersed cellstaken from original tissues, primary culture, or from a cell line orcell strain.

The term “coacervation” or phase separation as used herein refers to amacromolecular aggregation process brought about by partial desolvationof fully solvated macromolecules. A distinction is drawn between simpleand complex coacervation. In simple coacervation, there is only onecolloidal solute, and phase separation is induced by addition of alcoholor salt, change in temperature or change in pH. In complex coacervation,which deals with separations containing more than one solute, anoppositely charged substance is added to a polymer solution leading tothe formation of a coacervate phase via an anion-cation interaction.These phase separation processes can be used to encapsulate solid orliquid drug particles, which are dispersed in a polymer solution.

The term “complex” as used herein refers to an entity composed ofmolecules in which the constituents maintain much of their chemicalidentity.

The term “contact” and its various grammatical forms as used hereinrefers to a state or condition of touching or of immediate or localproximity.

The term “constitutional repeating unit” as used herein refers to thesmallest constitutional unit, the repetition of which constitutes aregular macromolecule (or oligomer molecule or block).

The term “constitutional unit” as used herein refers to an atom or groupof atoms in a macromolecule or oligomer molecule, comprising a part ofthe chain together with its pendant atoms or groups of atoms, if any.

The term “copolymer” as used herein refers to a polymer derived frommore than one species of monomer. Copolymers that are obtained bycopolymerization of two monomer species are sometimes termed bipolymers,those obtained from three monomers terpolymers, those obtained from fourmonomers quaterpolymers, etc.

The term “copolymerization” as used herein refers to polymerization inwhich a copolymer is formed.

The term “critical micelle temperature (CMT) also known as Krafft point”as used herein refers to a narrow temperature range above which thesolubility of a surfactant rises sharply. At this temperature, thesolubility of the surfactant becomes equal to the critical micelleconcentration. It is best determined, for example, by locating theabrupt change in slope of a graph of the logarithm of the solubilityagainst t or 1/T.

The term “crosslink” as used herein refers to a constitutional unitconnecting two parts of a macromolecule that were separate molecules oron distant parts of the same molecule. A network may be thought toconsist of many “primary chains” that are interconnected by a number ofcrosslinks. The crosslink can be a covalent bond, a site of weakerchemical interactions, a portion of crystallites, and even a physicalentanglement.

The term “diblock copolymer” as used herein refers to a polymerconsisting of two types of monomers, A and B. The monomers are arrangedsuch that there is a chain of each monomer, and those two chains aregrafted together to form a single copolymer chain.

The term “effective amount” as used herein refers to the amountnecessary or sufficient to realize a desired biologic effect.

The term “encapsulate” or “encapsulation” as used herein refers to aprocess in which tiny particles are enclosed inside a semipermeablemembrane, usually approximately spherical.

The term “fragment” or “peptide fragment” as used herein refers to asmall part derived, cut off, or broken from a larger peptide,polypeptide or protein, which retains the desired biological activity ofthe larger peptide, polypeptide or protein.

The terms “functional equivalent” or “functionally equivalent” are usedinterchangeably herein to refer to substances, molecules,polynucleotides, proteins, peptides, or polypeptides having similar oridentical effects. The “hemoglobin functional equivalent” as used hereinrefers to a molecule, a compound or a complex that appropriately storesand releases oxygen in accordance with an oxygen dissociation curve.

The term “fusion protein” as used herein refers to a protein orpolypeptide constructed by combining multiple protein domains orpolypeptides for the purpose of creating a single polypeptide or proteinwith functional properties derived from each of the original proteins orpolypeptides. Creation of a fusion protein may be accomplished byoperatively ligating or linking two different nucleotides sequences thatencode each protein domain or polypeptide via recombinant DNAtechnology, thereby creating a new polynucleotide sequences that codesfor the desired fusion protein. Alternatively, a fusion protein maybecreated by chemically joining the desired protein domains.

The term “gene cassette” is a type of mobile genetic element or cassettethat contains a gene of interest and a recombination site. The gene mayexist incorporated into an integron or freely as circular DNA. Genecassettes often carry antibiotic resistance genes. The cassette is apre-existing structure into which an insert can be moved. A geneconversion process occurs in which the old gene is replaced with a copyof a silent gene and the new copy becomes active. As the processinvolves replacing one ready-made construct with another in an activeslot, it is termed a cassette mechanism.

The term “genetic engineering” or “genetically engineered” as usedherein refers to the manipulation of DNA to produce new types oforganisms, usually by inserting or deleting genes.

The term “hemorrhagic shock” as used herein refers to a condition ofreduced tissue perfusion, resulting in the inadequate delivery of oxygenand nutrients that are necessary for cellular function. Whenevercellular oxygen demand outweighs supply, both the cell and the organismare in a state of shock. On a multicellular level, the definition ofshock becomes more difficult because not all tissues and organs willexperience the same amount of oxygen imbalance for a given clinicaldisturbance. The 4 classes of shock, are Hypovolemic, Vasogenic(septic), Cardiogenic, and Neurogenic. (Blalock A. Principle of SurgicalCare, Shock, and Other Problems. St Louis: Mosby; 1940.) Hypovolemicshock, the most common type, results from a loss of circulating bloodvolume from clinical etiologies, such as penetrating and blunt trauma,gastrointestinal bleeding, and obstetrical bleeding. Hemorrhagic shockproduced by rapid and significant loss of intravascular volume may leadsequentially to hemodynamic instability, decreases in oxygen delivery,decreased tissue perfusion, cellular hypoxia, organ damage, and death.

The term “hybridization” refers to the process of combiningcomplementary, single-stranded nucleic acids into a single molecule.Nucleotides will bind to their complement under normal conditions, sotwo perfectly complementary strands will bind (or ‘anneal’) to eachother readily. However, due to the different molecular geometries of thenucleotides, a single inconsistency between the two strands will makebinding between them more energetically unfavorable. Measuring theeffects of base incompatibility by quantifying the rate at which twostrands anneal can provide information as to the similarity in basesequence between the two strands being annealed. The term “specificallyhybridizes” as used herein refers to the process whereby a nucleic aciddistinctively or definitively forms base pairs with complementaryregions of at least one strand of DNA that was not originally paired tothe nucleic acid. For example, a nucleic acid that may bind or hybridizeto at least a portion of an mRNA of a cell encoding a peptide comprisinga specific protein sequence may be considered a nucleic acid thatspecifically hybridizes. A nucleic acid that selectively hybridizesundergoes hybridization, under stringent hybridization conditions, ofthe nucleic acid sequence to a specified nucleic acid target sequence toa detectably greater degree (e.g., at least 2-fold over background) thanits hybridization to non-target nucleic acid sequences and to thesubstantial exclusion of non-target nucleic acids. Selectivelyhybridizing sequences typically have about at least 80% sequenceidentity, at least 90% sequence identity, or at least 100% sequenceidentity (i.e., complementary) with each other.

The term “hydrogel” as used herein refers to gel in which the swellingagent is water. The network component of a hydrogel is usually a polymernetwork.

The term “liposome” as used herein refers to an artificially formedsingle or multi-layer spherical lipid bilayer structure, for example,made from solution of lipids in organic solvents dispersed in aqueousmedia.

The term “micelle” as used herein refers to an electrically chargedcolloidal particle, usually organic in nature, in which all of thehydrophobic portions of the molecule are inwardly directed, leaving thehydrophilic portions in contact with the surrounding aqueous phase. Ifthe major phase is hydrophobic, the inverse arrangement will be found.

The term “molecule” as used herein refers to a chemical unit composed ofone or more atoms.

The term “monomer” as used herein refers to a substance, each of themolecules of which can, on polymerization, contribute one or moreconstitutional units in the structure of the macromolecule.

The term “mutation” as used herein refers to a change of the DNAsequence within a gene or chromosome of an organism resulting in thecreation of a new character or trait not found in the parental type, orthe process by which such a change occurs in a chromosome, eitherthrough an alteration in the nucleotide sequence of the DNA coding for agene or through a change in the physical arrangement of a chromosome.Three mechanisms of mutation include substitution (exchange of one basepair for another), addition (the insertion of one or more bases into asequence), and deletion (loss of one or more base pairs).

The term “nanocarrier” as used herein refers to a nanomaterial beingused as a transport module for another substance, such as a drug.Commonly used nanocarriers include micelles, polymers, carbon-basedmaterials, liposomes and other substances.

The term “nanoparticle” or nanomaterial as used herein refers to aparticle or material with at least one dimension of 1×10-9 m-999×10-9 m.

The term “natural polymer” as used herein refers to a polymer derivedfrom biological systems, including, without limitation, a protein, DNA,RNA and polysaccharides.

The term “nucleic acid” as used herein to refer to a deoxyribonucleotideor ribonucleotide polymer in either single- or double-stranded form, andunless otherwise limited, encompasses known analogues having theessential nature of natural nucleotides in that they hybridize tosingle-stranded nucleic acids in a manner similar to naturally occurringnucleotides (e.g., peptide nucleic acids).

The term “nucleotide” as used herein to refer to a chemical compoundthat consists of a heterocyclic base, a sugar, and one or more phosphategroups. In the most common nucleotides, the base is a derivative ofpurine or pyrimidine, and the sugar is the pentose deoxyribose orribose. Nucleotides are the monomers of nucleic acids, with three ormore bonding together in order to form a nucleic acid. Nucleotides arethe structural units of RNA, DNA, and several cofactors, including, butnot limited to, CoA, FAD, DMN, NAD, and NADP. Purines include adenine(A), and guanine (G); pyrimidines include cytosine (C), thymine (T), anduracil (U).

The phrase “operatively linked” as used herein refers to a linkage inwhich two or more protein domains or polypeptides are ligated orcombined via recombinant DNA technology or chemical reaction such thateach protein domain or polypeptide of the resulting fusion proteinretains its original function.

The term “P50” as used herein refers to the partial pressure of oxygen(P02) at which hemoglobin becomes 50% saturated with oxygen.

The term “parenteral” as used herein refers to introduction into thebody by way of an injection (i.e., administration by injection),including, for example, subcutaneously (i.e., an injection beneath theskin), intramuscularly (i.e., an injection into a muscle); intravenously(i.e., an injection into a vein), intrathecally (i.e., an injection intothe space around the spinal cord or under the arachnoid membrane of thebrain), intrasternal injection, or infusion techniques. A parenterallyadministered composition of the present invention is delivered using aneedle, e.g., a surgical needle. The term “surgical needle” as usedherein, refers to any needle adapted for delivery of fluid (i.e.,capable of flow) compositions of the present invention into a selectedanatomical structure.

The term “particle” as used herein refers to an extremely smallconstituent (e.g., nanoparticles, microparticles, or in some instanceslarger).

The term “peptide” is used herein to refer to two or more amino acidsjoined by a peptide bond.

The term “pharmaceutically acceptable salt” as used herein refers tothose salts, which are, within the scope of sound medical judgment,suitable for use in contact with the tissues of humans and lower animalswithout undue toxicity, irritation, allergic response and the like andare commensurate with a reasonable benefit/risk ratio. When used inmedicine the salts should be pharmaceutically acceptable, butnon-pharmaceutically acceptable salts may conveniently be used toprepare pharmaceutically acceptable salts thereof. Such salts include,but are not limited to, those prepared from the following acids:hydrochloric, hydrobromic, sulphuric, nitric, phosphoric, maleic,acetic, salicylic, p-toluene sulphonic, tartaric, citric, methanesulphonic, formic, malonic, succinic, naphthalene-2-sulphonic, andbenzene sulphonic. Also, such salts may be prepared as alkaline metal oralkaline earth salts, such as sodium, potassium or calcium salts of thecarboxylic acid group. By “pharmaceutically acceptable salt” is meantthose salts, which are, within the scope of sound medical judgment,suitable for use in contact with the tissues of humans and lower animalswithout undue toxicity, irritation, allergic response and the like andare commensurate with a reasonable benefit/risk ratio. Pharmaceuticallyacceptable salts are well-known in the art. For example, P. H. Stahl, etal. describe pharmaceutically acceptable salts in detail in “Handbook ofPharmaceutical Salts: Properties, Selection, and Use” (Wiley VCH,Zurich, Switzerland: 2002). The salts may be prepared in situ during thefinal isolation and purification of the compounds described within thepresent invention or separately by reacting a free base function with asuitable organic acid. Representative acid addition salts include, butare not limited to, acetate, adipate, alginate, citrate, aspartate,benzoate, benzenesulfonate, bisulfate, butyrate, camphorate,camphorsufonate, digluconate, glycerophosphate, hemisulfate, heptanoate,hexanoate, fumarate, hydrochloride, hydrobromide, hydroiodide,2-hydroxyethansulfonate(isethionate), lactate, maleate,methanesulfonate, nicotinate, 2-naphthalenesulfonate, oxalate, pamoate,pectinate, persulfate, 3-phenylpropionate, picrate, pivalate,propionate, succinate, tartrate, thiocyanate, phosphate, glutamate,bicarbonate, p-toluenesulfonate and undecanoate. Also, the basicnitrogen-containing groups may be quaternized with such agents as loweralkyl halides such as methyl, ethyl, propyl, and butyl chlorides,bromides and iodides; dialkyl sulfates like dimethyl, diethyl, dibutyland diamyl sulfates; long chain halides such as decyl, lauryl, myristyland stearyl chlorides, bromides and iodides; arylalkyl halides likebenzyl and phenethyl bromides and others. Water or oil-soluble ordispersible products are thereby obtained. Examples of acids, which maybe employed to form pharmaceutically acceptable acid addition saltsinclude such inorganic acids as hydrochloric acid, hydrobromic acid,sulphuric acid and phosphoric acid and such organic acids as oxalicacid, maleic acid, succinic acid and citric acid. Basic addition saltsmay be prepared in situ during the final isolation and purification ofcompounds described within the invention by reacting a carboxylicacid-containing moiety with a suitable base such as the hydroxide,carbonate or bicarbonate of a pharmaceutically acceptable metal cationor with ammonia or an organic primary, secondary or tertiary amine.Pharmaceutically acceptable salts include, but are not limited to,cations based on alkali metals or alkaline earth metals such as lithium,sodium, potassium, calcium, magnesium and aluminum salts and the likeand nontoxic quaternary ammonia and amine cations including ammonium,tetramethylammonium, tetraethylammonium, methylamine, dimethylamine,trimethylamine, triethylamine, diethylamine, ethylamine and the like.Other representative organic amines useful for the formation of baseaddition salts include ethylenediamine, ethanolamine, diethanolamine,piperidine, piperazine and the like. Pharmaceutically acceptable saltsalso may be obtained using standard procedures well known in the art,for example by reacting a sufficiently basic compound such as an aminewith a suitable acid affording a physiologically acceptable anion.Alkali metal (for example, sodium, potassium or lithium) or alkalineearth metal (for example calcium or magnesium) salts of carboxylic acidsmay also be made.

The term “pharmaceutical composition” as used herein refers to acomposition that is employed to prevent, reduce in intensity, cure orotherwise treat a target condition or disease.

The term “polymer” as used herein refers to any of various chemicalcompounds made of smaller, identical molecules (called monomers) linkedtogether. Polymers generally have high molecular weights. The process bywhich molecules are linked together to form polymers is called“polymerization.”

The term “polynucleotide” refers to a deoxyribopolynucleotide,ribopolynucleotide, or analogs thereof that have the essential nature ofa natural ribonucleotide in that they hybridize, under stringenthybridization conditions, to substantially the same nucleotide sequenceas naturally occurring nucleotides and/or allow translation into thesame amino acid(s) as the naturally occurring nucleotide(s). Apolynucleotide may be full-length or a subsequence of a native orheterologous structural or regulatory gene. Unless otherwise indicated,the term includes reference to the specified sequence as well as thecomplementary sequence thereof. Thus, DNAs or RNAs with backbonesmodified for stability or for other reasons are “polynucleotides” asthat term is intended herein. Moreover, DNAs or RNAs comprising unusualbases, such as inosine, or modified bases, such as tritylated bases, toname just two examples, are polynucleotides as the term is used herein.It will be appreciated that a great variety of modifications have beenmade to DNA and RNA that serve many useful purposes known to those ofskill in the art. The term polynucleotide as it is employed hereinembraces such chemically, enzymatically or metabolically modified formsof polynucleotides, as well as the chemical forms of DNA and RNAcharacteristic of viruses and cells, including among other things,simple and complex cells.

The term “protein” is used herein to refer to a large complex moleculeor polypeptide composed of amino acids. The sequence of the amino acidsin the protein is determined by the sequence of the bases in the nucleicacid sequence that encodes it.

The term “polypeptide” is used herein in its broadest sense to refer toa sequence of subunit amino acids, amino acid analogs orpeptidomimetics, wherein the subunits are linked by peptide bonds.

The terms “peptide”, “polypeptide” and “protein” also apply to aminoacid polymers in which one or more amino acid residue is an artificialchemical analogue of a corresponding naturally occurring amino acid, aswell as to naturally occurring amino acid polymers. The essential natureof such analogues of naturally occurring amino acids is that, whenincorporated into a protein that protein is specifically reactive toantibodies elicited to the same protein but consisting entirely ofnaturally occurring amino acids. The terms “polypeptide”, “peptide” and“protein” also are inclusive of modifications including, but not limitedto, glycosylation, lipid attachment, sulfation, gamma-carboxylation ofglutamic acid residues, hydroxylation and ADP-ribosylation. It will beappreciated, as is well known and as noted above, that polypeptides maynot be entirely linear. For instance, polypeptides may be branched as aresult of ubiquitination, and they may be circular, with or withoutbranching, generally as a result of posttranslational events, includingnatural processing event and events brought about by human manipulation,which do not occur naturally. Circular, branched and branched circularpolypeptides may be synthesized by non-translation natural process andby entirely synthetic methods, as well.

The term “phase” as used herein refers to a distinct state of matter ina system in which matter that is identical in chemical composition andphysical state and separated from other material by the phase boundary.

The term “recombinant proteins” as used herein refers to proteins thatcan result from the expression of recombinant DNA within living cellsare termed recombinant proteins.

The term “shock” as used herein refers to a state of inadequateperfusion, which does not sustain the physiologic needs of organtissues. Many conditions, including blood loss but also includingnonhemorrhagic states such as dehydration, sepsis, impairedautoregulation, obstruction, decreased myocardial function, and loss ofautonomic tone, may produce shock or shocklike states.

The term “solution” as used herein refers to a homogeneous mixture oftwo or more substances. It is frequently, though not necessarily, aliquid. In a solution, the molecules of the solute (or dissolvedsubstance) are uniformly distributed among those of the solvent.

The term “solvate” as used herein refers to a complex formed by theattachment of solvent molecules to that of a solute.

The term “solvent” as used herein refers to a substance capable ofdissolving another substance (termed a “solute”) to form a uniformlydispersed mixture (solution).

The phrase “subject” as used herein refers to a patient that (i) will beadministered at least pharmaceutical composition of the describedinvention, (ii) is receiving at least pharmaceutical composition of thedescribed invention; or (iii) has received at least one pharmaceuticalcomposition of the described invention, unless the context and usage ofthe phrase indicates otherwise.

The term “therapeutic agent” as used herein refers to a drug, molecule,nucleic acid, protein, composition or other substance that provides atherapeutic effect. The term “active” as used herein refers to theingredient, component or constituent of the compositions of the presentinvention responsible for the intended therapeutic effect. The terms“therapeutic agent” and “active agent” are used interchangeably herein.

The terms “therapeutically effective amount”, or “effective amount” oran “amount effective”, or “pharmaceutically effective amount” are usedinterchangeably to refer to an amount that is sufficient to provide theintended benefit of treatment. An effective amount of an active agentthat can be employed according to the described invention generallyranges from about 50 mg/kg body weight to about 1.5 g/kg body weight.However, dosage levels are based on a variety of factors, including thetype of injury, the age, weight, sex, medical condition of the patient,the severity of the condition, the route of administration, and theparticular active agent employed. Thus the dosage regimen may varywidely, but can be determined routinely by a physician using standardmethods. Additionally, the terms “therapeutically effective amount”,“amount effective” and “pharmaceutically effective amount” includeprophylactic or preventative amounts of the compositions of thedescribed invention. In prophylactic or preventative applications of thedescribed invention, pharmaceutical compositions or medicaments areadministered to a patient susceptible to, or otherwise at risk of, adisease, disorder or condition in an amount sufficient to eliminate orreduce the risk, lessen the severity, or delay the onset of the disease,disorder or condition, including biochemical, histologic and/orbehavioral symptoms of the disease, disorder or condition, itscomplications, and intermediate pathological phenotypes presentingduring development of the disease, disorder or condition. It ispreferred generally that a maximum dose be used, that is, the highestsafe dose according to some medical judgment. “Dose” and “dosage” areused interchangeably herein.

The term “treat” or “treating” includes abrogating, substantiallyinhibiting, slowing or reversing the progression of a disease, conditionor disorder, substantially ameliorating clinical or esthetical symptomsof a condition, substantially preventing the appearance of clinical oresthetical symptoms of a disease, condition, or disorder, and protectingfrom harmful or annoying symptoms. Treating further refers toaccomplishing one or more of the following: (a) reducing the severity ofthe disorder; (b) limiting development of symptoms characteristic of thedisorder(s) being treated; (c) limiting worsening of symptomscharacteristic of the disorder(s) being treated; (d) limiting recurrenceof the disorder(s) in patients that have previously had the disorder(s);and (e) limiting recurrence of symptoms in patients that were previouslyasymptomatic for the disorder(s).

The term “Tropoelastin” as used herein refers to a protein that isexpressed and post-translationally modified from the gene encodingelastin, prior to cross-linking to form elastin. Martin et al. (1995)Gene, 154, 159-166, details the making of the synthetic gene andsubsequent expression of synthetic human elastin (SHEL). A used herein,“tropoelastin” encompasses full length tropoelastin, isoforms oftropoelastin, genetically engineered tropoelastin constructs, andfragments and derivatives of tropoelastin.

The term “transition temperature (for liquid crystals)” as used hereinrefers to the temperature at which the transition from mesophase X tomesophase Y occurs. A mesophase is a phase occurring over a definiterange of temperature, pressure, or concentration within a mesomorphicstate. A mesomorphic state of matter is one in which the degree ofmolecular order is intermediate between the perfect three-dimensional,long-range positional and orientational order found in solid crystalsand the absence of long-range order found in isotropic liquids, gases,and amorphous solids.

The terms “variants”, “mutants”, and “derivatives” are used herein torefer to sequences with substantial identity to a reference sequence. Askilled artisan can produce polypeptide variants having single ormultiple amino acid substitutions, deletions, additions or replacements.These variants may include inter alia: (a) variants in which one or moreamino acid residues are substituted with conservative ornon-conservative amino acids; (b) variants in which one or more aminoacids are added; (c) variants in which at least one amino acid includesa substituent group; (d) variants in which amino acid residues from onespecies are substituted for the corresponding residue in anotherspecies, either at conserved or non-conserved positions; and (d)variants in which a target protein is fused with another peptide orpolypeptide such as a fusion partner, a protein tag or other chemicalmoiety, that may confer useful properties to the target protein, suchas, for example, an epitope for an antibody. The techniques forobtaining such variants, including genetic (suppressions, deletions,mutations, etc.), chemical, and enzymatic techniques are known.

According to one aspect, the described invention provides apharmaceutical composition comprising a polymer and a hemoglobin orfunctional equivalent thereof, wherein the pharmaceutical composition iseffective to deliver oxygen, prevent or treat conditions caused by bloodloss or anemia or other blood disorder, and improve subject survival.

According to one embodiment, The composition is prepared by bringinginto association or contact a protein polymer and a hemoglobin,subunit(s), fragment(s), derivatives(s) or functional equivalent thereofor a pharmaceutically acceptable salt or solvate thereof (“activecompound”) with a carrier which constitutes one or more accessoryagents. In general, the formulations are prepared by uniformly andintimately bringing into association the active agent(s) with liquidcarriers or finely divided solid carriers or both and then, ifnecessary, shaping the product into the desired formulation.

According to one embodiment of the described invention, the conditioncaused by blood loss includes, without limitation, hemorrhagic shock.

According to one embodiment of the described invention, the polymer is anatural polymer, a synthetic polymer (including degradable andnon-degradable), a hybrid polymer, or a recombinant polymer.

Exemplary synthetic degradable polymers include, without limitation,poly(c-caprolactone) (PCL), poly(ε-caprolactone-co-ethyl ethylenephosphate) (PCLEEP), poly(lactic acid) (PLA), poly(lactic-co-glycolicacid) (PLGA), poly(lactic acid-co-ε-caprolactone) (PLACL), andpolydioxanone (PDO).

According to one embodiment, the polymer is a protein polymer, apolynucleotide polymer (e.g. a DNA, or an RNA), a polysaccharidepolymer, or a synthetic polymer.

According to one embodiment, the protein polymer is a natural proteinpolymer, a synthetic protein polymer, or a recombinant protein polymer.

According to one embodiment, the hemoglobin is natural, synthetic,recombinant, a fragment, a subunit, or a derivative.

According to one embodiment, a hemoglobin functional equivalentincludes, without limitation, a molecule comprising an affinity foroxygen.

According to one embodiment, the hemoglobin, subunit(s), fragment(s),derivative(s), or functional equivalent thereof can be formulated per seor in salt form.

According to one embodiment, the hemoglobin or subunit(s) or fragment(s)or derivative(s) or functional equivalent thereof can be truncated ormodified.

According to one embodiment, the polymer binds to the hemoglobin,subunit(s), fragment(s), derivative(s), or functional equivalent thereofvia a covalent bond, an ionic bond, a hydrogen bond, a hydrophobicforce, encapsulation, or is operatively linked via fusion.

According to one embodiment, the polymer contacts the hemoglobin,subunit(s), fragment(s), derivative(s), or functional equivalentthereof.

According to one embodiment, the polymer is operatively linked to ahemoglobin subunit(s) or fragment(s) thereof.

According to one embodiment, the polymer binds to hemoglobin via achemical reaction.

According to another embodiment, the polymer and hemoglobin comprise afusion protein.

According to one embodiment, the protein polymer is an elastin-likeprotein (ELP), a silk-like protein (SLP), or a silk-elastin like protein(SELP).

According to one embodiment, the ELP comprises a pentameric amino acidmotif of (Val-Pro-Gly-Xaa-Gly)n, wherein Xaa specifies any amino acidand n denotes the number of repetitive motifs.

According to some such embodiments, n is at least 20, 25, 30, 35, 40,45, 50, 55, 60, 65, 70, 75, 80, 85, 90. According to some embodiments, nranges from 20-30, 20-40, 20-50, 20-60, 20-70, 20-80, 20-90, 30-40,30-50, 30-60, 30-70, 30-80, 30-90, 40-50, 40-60, 40-70, 40-80, 40-90,50-60, 50-70, 50-80, 50-90, 60-70, 60-80, 60-90, 70-80, 70-90, or 80-90.

According to one embodiment, the ELP can be formulated per se or in saltform.

According to one embodiment, the ELP can be truncated, modified, orderivatized.

According to one embodiment of the described invention, the ELP is adiblock copolymer, comprising a hydrophobic block of amino acids and ahydrophilic block of amino acids, which can assemble into a sphericalnanoparticle above a critical micelle temperature (CMT) to encapsulatethe hemoglobin, a subunit(s), a fragment(s), a derivative(s), or afunctional equivalent thereof at its core.

According to one embodiment, the hydrophilic block of the ELP diblockcopolymer comprises a pentameric amino acid motif of(Val-Pro-Gly-Xaa-Gly)n, wherein n=20-90, and Xaa is a hydrophilic aminoacid, for example, lysine (+), arginine (+), aspartate (−), glutamate(−), serine, threonine, asparagine, glutamine, and histidine.

According to one embodiment, the hydrophilic block of the ELP diblockcopolymer comprises a pentameric amino acid motif of(Val-Pro-Gly-Xaa-Gly)n, wherein n=20-90, and Xaa is Ser or aconservative amino acid substitute thereof, for example, Thr.

According to one embodiment, the hydrophilic block of the ELP diblockcopolymer comprises a pentameric amino acid motif of(Val-Pro-Gly-Xaa-Gly)n, wherein n=40-60.

According to one embodiment, the hydrophobic block of the ELP diblockcopolymer comprises a pentameric amino acid motif of(Val-Pro-Gly-Ser-Gly)n, wherein n=20-90, and Xaa is a hydrophobic aminoacid, for example, alanine, valine, leucine, isoleucine, proline,phenylalanine, tryptophan, and methionine.

According to one embodiment, the hydrophobic block of the ELP diblockcopolymer comprises a pentameric amino acid motif of(Val-Pro-Gly-Xaa-Gly)n, wherein n=20-90, and Xaa is Ile or aconservative amino acid substitute thereof, for example, Leu or Met orVal.

According to one embodiment, the hydrophobic block of the ELP diblockcopolymer comprises a pentameric amino acid motif of(Val-Pro-Gly-Xaa-Gly)n, wherein n=40-60.

According to one embodiment of the described invention, the hemoglobinsubunit or fragment is operatively linked to the hydrophobic block ofthe ELP to facilitate enclosing of hemoglobin or functional equivalentthereof within the core of an ELP nanoparticle.

According to one embodiment, the hemoglobin, subunit(s), fragment(s),derivative(s), or functional equivalent thereof is operatively linked tothe C-terminus of the ELP.

According to one embodiment, the hemoglobin, subunit(s), fragment(s),derivative(s), or functional equivalent thereof is operatively linked toa hydrophobic block of the ELP.

According to one embodiment, the hemoglobin, subunit(s), fragment(s),derivative(s), or functional equivalent thereof is operatively linked tothe hydrophobic block of the ELP via a chemical reaction.

According to another embodiment, the hemoglobin, subunit(s),fragment(s), derivative(s), or functional equivalent thereof that isoperatively linked to the hydrophobic block of the ELP comprises afusion protein.

According to one embodiment, the specific polynucleotide is contained ina vector and/or host cell.

According to one embodiment, the fusion protein is encoded by apolynucleotide comprising a nucleotide sequence that encodes arecombinant ELP operatively linked to a nucleotide sequence that encodesa hemoglobin, a subunit, a fragment, or a functional equivalent thereof.

According to one embodiment, the polynucleotide sequence that encodesthe hemoglobin, a subunit, a fragment, or a functional equivalentthereof is selected from the group consisting of SEQ ID NO. 1, SEQ IDNO. 2 and SEQ ID NO. 3.

According to one embodiment, a hemoglobin, a subunit, a fragment, or afunctional equivalent thereof comprises an amino acid sequence selectedfrom the group consisting of: SEQ ID NO. 4, SEQ ID NO. 5 and SEQ ID NO.6.

According to one embodiment, the ELP comprises amino acid sequence SEQID NO. 7.

According to one embodiment, the fusion protein comprises an ELP ofamino acid sequence SEQ ID NO. 7 operatively linked to one or morehemoglobin subunits of amino acid sequences selected from the groupconsisting of SEQ ID NO. 4, SEQ ID NO. 5 and SEQ ID NO. 6.

According to one embodiment, the fusion protein comprises amino acidsequence SEQ ID NO. 8 containing an ELP of amino acid sequence SEQ IDNO. 7 operatively linked to a hemoglobin subunit of amino acid sequenceSEQ ID NO. 4.

According to one embodiment, the fusion protein comprises amino acidsequence SEQ ID NO. 9 containing an ELP of amino acid sequence SEQ IDNO. 7 operatively linked to a hemoglobin subunit of amino acid sequenceSEQ ID NO. 5.

According to one embodiment, the fusion protein comprises amino acidsequence SEQ ID NO. 10 containing an ELP of amino acid sequence SEQ IDNO. 7 operatively linked to a hemoglobin subunit of amino acid sequenceSEQ ID NO. 6.

According to one embodiment of the described invention, thepharmaceutical composition comprises a therapeutic amount of a proteinpolymer-encapsulated hemoglobin molecule.

According to one embodiment, the protein polymer-encapsulated hemoglobinmolecule is a fusion protein consisting essentially of a protein polymeroperatively linked to a hemoglobin subunit or a fragment, wherein thehemoglobin is encapsulated within the protein polymer.

According to one embodiment, the pharmaceutical composition furthercomprises one or more pharmaceutically acceptable carriers.

According to another aspect, the described invention provides a methodfor treating a condition due to blood loss, and improving subjectsurvival, the method comprising: (1) administering a biocompatiblepharmaceutical composition comprising a therapeutic amount of a complexcomprising a polymer associated with a hemoglobin, a subunit(s), afragment(s), a derivative(s), or a functional equivalent thereof.

According to one embodiment of the described invention, the conditioncaused by blood loss includes, without limitation, hemorrhagic shock.

According to one embodiment of the described invention, the polymer is anatural polymer, a synthetic polymer (including degradable andnon-degradable), a hybrid polymer, or a recombinant polymer.

Exemplary synthetic degradable polymers include, without limitation,poly(c-caprolactone) (PCL), poly(ξ-caprolactone-co-ethyl ethylenephosphate) (PCLEEP), poly(lactic acid) (PLA), poly(lactic-co-glycolicacid) (PLGA), poly(lactic acid-co-ε-caprolactone) (PLACL), andpolydioxanone (PDO).

According to one embodiment, the polymer is a protein polymer, apolynucleotide polymer (e.g. a DNA, or an RNA), a polysaccharidepolymer, or a synthetic polymer.

According to one embodiment, the protein polymer is a natural proteinpolymer, a synthetic protein polymer, or a recombinant protein polymer.

According to one embodiment, the hemoglobin is natural, synthetic,recombinant, a fragment, a subunit, or a derivative.

According to one embodiment, a hemoglobin functional equivalentincludes, without limitation, a molecule comprising an affinity foroxygen.

According to one embodiment, the hemoglobin, subunit(s), fragment(s),derivative(s), or functional equivalent thereof can be formulated per seor in salt form.

According to one embodiment, the hemoglobin or subunit(s) or fragment(s)or derivative(s) or functional equivalent thereof can be truncated ormodified.

According to one embodiment, the polymer binds to the hemoglobin,subunit(s), fragment(s), derivative(s), or functional equivalent thereofvia a covalent bond, an ionic bond, a hydrogen bond, a hydrophobicforce, encapsulation, or is operatively linked via fusion.

According to one embodiment, the polymer contacts the hemoglobin,subunit(s), fragment(s), derivative(s), or functional equivalentthereof.

According to one embodiment, the polymer is operatively linked to ahemoglobin subunit(s) or fragment(s) thereof.

According to one embodiment, the polymer binds to hemoglobin via achemical reaction.

According to another embodiment, the polymer and hemoglobin comprise afusion protein.

According to one embodiment, the protein polymer is an elastin-likeprotein (ELP), a silk-like protein (SLP), or a silk-elastin like protein(SELP).

According to one embodiment, the ELP comprises a pentameric amino acidmotif of (Val-Pro-Gly-Xaa-Gly)n, wherein Xaa specifies any amino acidand n denotes the number of repetitive motifs.

According to some such embodiments, n is at least 20, 25, 30, 35, 40,45, 50, 55, 60, 65, 70, 75, 80, 85, 90. According to some embodiments, nranges from 20-30, 20-40, 20-50, 20-60, 20-70, 20-80, 20-90, 30-40,30-50, 30-60, 30-70, 30-80, 30-90, 40-50, 40-60, 40-70, 40-80, 40-90,50-60, 50-70, 50-80, 50-90, 60-70, 60-80, 60-90, 70-80, 70-90, or 80-90.

According to one embodiment, the ELP can be formulated per se or in saltform.

According to one embodiment, the ELP can be truncated, modified, orderivatized.

According to one embodiment of the described invention, the ELP is adiblock copolymer, comprising a hydrophobic block of amino acids and ahydrophilic block of amino acids, which can assemble into a sphericalnanoparticle above a critical micelle temperature (CMT) to encapsulatethe hemoglobin, a subunit(s), a fragment(s), a derivative(s), or afunctional equivalent thereof at its core.

According to one embodiment, the hydrophilic block of the ELP diblockcopolymer comprises a pentameric amino acid motif of(Val-Pro-Gly-Xaa-Gly)n, wherein n=20-90, and Xaa is a hydrophilic aminoacid, for example, lysine (+), arginine (+), aspartate (−), glutamate(−), serine, threonine, asparagine, glutamine, and histidine.

According to one embodiment, the hydrophilic block of the ELP diblockcopolymer comprises a pentameric amino acid motif of(Val-Pro-Gly-Xaa-Gly)n, wherein n=20-90, and Xaa is Ser or aconservative amino acid substitute thereof, for example, Thr.

According to one embodiment, the hydrophilic block of the ELP diblockcopolymer comprises a pentameric amino acid motif of(Val-Pro-Gly-Xaa-Gly)n, wherein n=40-60.

According to one embodiment, the hydrophobic block of the ELP diblockcopolymer comprises a pentameric amino acid motif of(Val-Pro-Gly-Ser-Gly)n, wherein n=20-90, and Xaa is a hydrophobic aminoacid, for example, alanine, valine, leucine, isoleucine, proline,phenylalanine, tryptophan, and methionine.

According to one embodiment, the hydrophobic block of the ELP diblockcopolymer comprises a pentameric amino acid motif of(Val-Pro-Gly-Xaa-Gly)n, wherein n=20-90, and Xaa is Ile or aconservative amino acid substitute thereof, for example, Leu or Met orVal.

According to one embodiment, the hydrophobic block of the ELP diblockcopolymer comprises a pentameric amino acid motif of(Val-Pro-Gly-Xaa-Gly)n, wherein n=40-60.

According to one embodiment of the described invention, the hemoglobinsubunit or fragment is operatively linked to the hydrophobic block ofthe ELP to facilitate enclosing of hemoglobin or functional equivalentthereof within the core of an ELP nanoparticle.

According to one embodiment, the hemoglobin, subunit(s), fragment(s),derivative(s), or functional equivalent thereof is operatively linked tothe C-terminus of the ELP.

According to one embodiment, the hemoglobin, subunit(s), fragment(s),derivative(s), or functional equivalent thereof is operatively linked toa hydrophobic block of the ELP.

According to one embodiment, the hemoglobin, subunit(s), fragment(s),derivative(s), or functional equivalent thereof is operatively linked tothe hydrophobic block of the ELP via a chemical reaction.

According to another embodiment, the hemoglobin, subunit(s),fragment(s), derivative(s), or functional equivalent thereof that isoperatively linked to the hydrophobic block of the ELP comprises afusion protein.

According to one embodiment, the specific polynucleotide is contained ina vector and/or host cell.

According to one embodiment, the fusion protein is encoded by apolynucleotide comprising a nucleotide sequence that encodes arecombinant ELP operatively linked to nucleotide sequence that encodes ahemoglobin, a subunit, a fragment, or a functional equivalent thereof.

According to one embodiment, the polynucleotide sequence that encodesthe hemoglobin, a subunit, a fragment, or a functional equivalentthereof is selected from the group consisting of SEQ ID NO. 1, SEQ IDNO. 2 and SEQ ID NO. 3.

According to one embodiment, a hemoglobin, a subunit, a fragment, or afunctional equivalent thereof comprises an amino acid sequence selectedfrom the group consisting of: SEQ ID NO. 4, SEQ ID NO. 5 and SEQ ID NO.6.

According to one embodiment, the ELP comprises amino acid sequence SEQID NO. 7.

According to one embodiment, the fusion protein comprises an ELP ofamino acid sequence SEQ ID NO. 7 operatively linked to one or morehemoglobin subunits of amino acid sequences selected from the groupconsisting of SEQ ID NO. 4, SEQ ID NO. 5 and SEQ ID NO. 6.

According to one embodiment, the fusion protein comprises amino acidsequence SEQ ID NO. 8 containing an ELP of amino acid sequence SEQ IDNO. 7 operatively linked to a hemoglobin subunit of amino acid sequenceSEQ ID NO. 4.

According to one embodiment, the fusion protein comprises amino acidsequence SEQ ID NO. 9 containing an ELP of amino acid sequence SEQ IDNO. 7 operatively linked to a hemoglobin subunit of amino acid sequenceSEQ ID NO. 5.

According to one embodiment, the fusion protein comprises amino acidsequence SEQ ID NO. 10 containing an ELP of amino acid sequence SEQ IDNO. 7 operatively linked to a hemoglobin subunit of amino acid sequenceSEQ ID NO. 6.

According to one embodiment of the described invention, thepharmaceutical composition comprises a therapeutic amount of a proteinpolymer-encapsulated hemoglobin molecule.

According to one embodiment, the protein polymer-encapsulated hemoglobinmolecule is a fusion protein consisting essentially of a protein polymeroperatively linked to a hemoglobin subunit or a fragment, wherein thehemoglobin is encapsulated within the protein polymer.

According to one embodiment, the pharmaceutical composition furthercomprises one or more pharmaceutically acceptable carriers.

Formulations of pharmaceutical composition may be administered inpharmaceutically acceptable solutions, which may routinely containpharmaceutically acceptable concentrations of salt, buffering agents,preservatives, compatible carriers, adjuvants, and optionally othertherapeutic ingredients.

For use in therapy, the pharmaceutical composition may be administeredto a subject parenterally through, e.g. a needle, a cannula, a catheter,and the like.

Formulations for injection may be presented in unit dosage form, e.g.,in ampoules or in multi-dose containers, with an added preservative. Thecompositions may take such forms as suspensions, solutions or emulsionsin oily or aqueous vehicles, and may contain formulatory agents such assuspending, stabilizing and/or dispersing agents. Pharmaceuticalformulations for parenteral administration include aqueous solutions ofthe active compounds in water-soluble form. Additionally, suspensions ofthe active compounds may be prepared as appropriate oily injectionsuspensions. Suitable lipophilic solvents or vehicles include fatty oilssuch as sesame oil, or synthetic fatty acid esters, such as ethyl oleateor triglycerides, or liposomes. Aqueous injection suspensions maycontain substances, which increase the viscosity of the suspension, suchas sodium carboxymethyl cellulose, sorbitol, or dextran. Optionally, thesuspension may also contain suitable stabilizers or agents, whichincrease the solubility of the compounds to allow for the preparation ofhighly concentrated solutions. Alternatively, the active compounds maybe in powder form for constitution with a suitable vehicle, e.g.,sterile pyrogen-free water, before use.

The pharmaceutical compositions also may comprise suitable solid or gelphase carriers or excipients. Examples of such carriers or excipientsinclude, but are not limited to, calcium carbonate, calcium phosphate,various sugars, starches, cellulose derivatives, gelatin, and polymerssuch as polyethylene glycols.

The protein polymer complex may be administered per se (neat) or in theform of a pharmaceutically acceptable salt. When used in medicine thesalts should be pharmaceutically acceptable, but non-pharmaceuticallyacceptable salts may conveniently be used to prepare pharmaceuticallyacceptable salts thereof.

The formulations may be presented conveniently in unit dosage form andmay be prepared by any of the methods well known in the art of pharmacy.

The pharmaceutical protein polymer-hemoglobin complex or apharmaceutically acceptable salt, solvate or prodrug thereof may bemixed with other active materials that do not impair the desired action,or with materials that supplement the desired action.

Solutions or suspensions used for parenteral administration may include,but are not limited to, for example, the following components: a sterilediluent such as water for injection, saline solution, fixed oils,polyethylene glycols, glycerine, propylene glycol or other syntheticsolvents; antibacterial agents such as benzyl alcohol or methylparabens; antioxidants such as ascorbic acid or sodium bisulfite;chelating agents such as ethylenediaminetetraacetic acid; buffers suchas acetates, citrates or phosphates and agents for the adjustment oftonicity such as sodium chloride or dextrose. The parental preparationmay be enclosed in ampoules, disposable syringes or multiple dose vialsmade of glass or plastic. Administered intravenously, exemplary carriersare physiological saline or phosphate buffered saline (PBS).

Pharmaceutical compositions for parenteral administration comprisepharmaceutically acceptable sterile aqueous or nonaqueous solutions,dispersions, suspensions or emulsions and sterile powders forreconstitution into sterile injectable solutions or dispersions.Examples of suitable aqueous and nonaqueous carriers, diluents, solventsor vehicles include water, ethanol, polyols (propylene glycol,polyethylene glycol, glycerol, and the like), suitable mixtures thereof,vegetable oils (such as olive oil) and injectable organic esters such asethyl oleate. Proper fluidity may be maintained, for example, by the useof a coating such as lecithin, by the maintenance of the requiredparticle size in the case of dispersions, and by the use of surfactants.

These compositions may also contain adjuvants including preservativeagents, wetting agents, emulsifying agents, and dispersing agents.Prevention of the action of microorganisms may be ensured by variousantibacterial and antifungal agents, for example, parabens,chlorobutanol, phenol, sorbic acid, and the like. It may also bedesirable to include isotonic agents, for example, sugars, sodiumchloride and the like. Prolonged absorption of the injectablepharmaceutical form may be brought about by the use of agents delayingabsorption, for example, aluminum monostearate and gelatin.

Suspensions, in addition to the active compounds, may contain suspendingagents, as, for example, ethoxylated isostearyl alcohols,polyoxyethylene sorbitol and sorbitan esters, microcrystallinecellulose, aluminum metahydroxide, bentonite, agar-agar, tragacanth, andmixtures thereof.

The injectable formulations may be sterilized, for example, byfiltration through a bacterial-retaining filter or by incorporatingsterilizing agents in the form of sterile solid compositions that may bedissolved or dispersed in sterile water or other sterile injectablemedium just prior to use. Injectable preparations, for example, sterileinjectable aqueous or oleaginous suspensions may be formulated accordingto the known art using suitable dispersing or wetting agents andsuspending agents. The sterile injectable preparation also may be asterile injectable solution, suspension or emulsion in a nontoxic,parenterally acceptable diluent or solvent such as a solution in1,3-butanediol. Among the acceptable vehicles and solvents that may beemployed are water, Ringer's solution, U.S.P. and isotonic sodiumchloride solution. In addition, sterile, fixed oils conventionally areemployed or as a solvent or suspending medium. For this purpose anybland fixed oil may be employed including synthetic mono- ordiglycerides. In addition, fatty acids such as oleic acid are used inthe preparation of injectables.

Formulations for parenteral administration include aqueous andnon-aqueous sterile injection solutions that may contain anti-oxidants,buffers, bacteriostats and solutes, which render the formulationisotonic with the blood of the intended recipient; and aqueous andnon-aqueous sterile suspensions, which may include suspending agents andthickening agents. The formulations may be presented in unit-dose ormulti-dose containers, for example sealed ampules and vials, and may bestored in a freeze-dried (lyophilized) condition requiring only theaddition of the sterile liquid carrier, for example, saline,water-for-injection, immediately prior to use. Extemporaneous injectionsolutions and suspensions may be prepared from sterile powders, granulesand tablets of the kind previously described.

Suitable buffering agents include: acetic acid and a salt (1-2% w/v);citric acid and a salt (1-3% w/v); boric acid and a salt (0.5-2.5% w/v);and phosphoric acid and a salt (0.8-2% w/v). Suitable preservativesinclude benzalkonium chloride (0.003-0.03% w/v); chlorobutanol (0.3-0.9%w/v); parabens (0.01-0.25% w/v) and thimerosal (0.004-0.02% w/v).

The polymer-hemoglobin complex may be provided in particles. The term“particles” as used herein refers to nano or microparticles (or in someinstances larger) that may contain in whole or in part the hemoglobin orfunctional equivalent of hemoglobin as described herein. According toone embodiment, the particles can contain the hemoglobin, subunit(s),fragment(s), derivative(s), or functional equivalent thereof in a coresurrounded by the polymer. According to one embodiment, the therapeuticcomplex can be dispersed throughout the particles. According to oneembodiment, the therapeutic complex can be adsorbed into the particles.The particles can be of any order release kinetics, including zero orderrelease, first order release, second order release, delayed release,sustained release, immediate release, etc., and any combination thereof.The particle may include, in addition to the therapeutic complex, any ofthose protein polymers routinely used in the art of pharmacy andmedicine, including, but not limited to, erodible, nonerodible,biodegradable, or nonbiodegradable material or combinations thereof.According to one embodiment, the particles may be microcapsules ofprotein polymers that contain the hemoglobin, subunit(s), fragment(s),derivative(s), or functional equivalent thereof. According to oneembodiment, the particles may be of virtually any shape.

The compositions of the present invention may be in the form of asterile injectable aqueous or oleaginous suspension. Such injectablepreparations may be formulated using suitable dispersing or wettingagents and suspending agents.

The sterile injectable preparation also may be a sterile injectablesolution or suspension in a nontoxic parenterally acceptable diluent orsolvent, for example, as a solution in 1,3-butanediol. A solutiongenerally is considered as a homogeneous mixture of two or moresubstances; it is frequently, though not necessarily, a liquid. In asolution, the molecules of the solute (or dissolved substance) areuniformly distributed among those of the solvent. A suspension is adispersion (mixture) in which a finely-divided species is combined withanother species, with the former being so finely divided and mixed thatit doesn't rapidly settle out. In everyday life, the most commonsuspensions are those of solids in liquid water. Among the acceptablevehicles and solvents that may be employed are water, Ringer's solution,and isotonic sodium chloride solution. In addition, sterile, fixed oilsare conventionally employed as a solvent or suspending medium. Forparenteral application, particularly suitable vehicles consist ofsolutions, preferably oily or aqueous solutions, as well as suspensions,emulsions, or implants. Aqueous suspensions may contain substances whichincrease the viscosity of the suspension and include, for example,sodium carboxymethyl cellulose, sorbitol and/or dextran. Optionally, thesuspension may also contain stabilizers.

The amount of the pharmaceutically acceptable carrier is that amountneeded to provide the necessary stability, dispersibility, consistencyand bulking characteristics to ensure a uniform pulmonary delivery ofthe composition to a subject in need thereof. Numerically the amount maybe from about 0.05% w to about 99.95% w, depending on the activity ofthe drug being employed. According to one embodiment, about 5% w toabout 95% will be used. The carrier may be one or a combination of twoor more pharmaceutical excipients, but generally will be substantiallyfree of any “penetration enhancers.” Penetration enhancers are surfaceactive compounds which promote penetration of a drug through a mucosalmembrane or lining and are proposed for use in intranasal, intrarectal,and intravaginal drug formulations. Exemplary penetration enhancersinclude bile salts, e.g., taurocholate, glycocholate, and deoxycholate;fusidates, e.g., taurodehydrofusidate; and biocompatible detergents,e.g., Tweens, Laureth-9, and the like. The use of penetration enhancersin formulations for the lungs, however, is generally undesirable becausethe epithelial blood barrier in the lung can be adversely affected bysuch surface active compounds. The dry powder compositions of thepresent invention are readily absorbed in the lungs without the need toemploy penetration enhancers.

According to some embodiments, the compositions of the describedinvention may be formulated with an excipient, vehicle or carrierselected from solvents, suspending agents, binding agents, fillers,lubricants, disintegrants, and wetting agents/surfactants/solubilizingagents.

The carrier can be liquid or solid and is selected with the plannedmanner of administration in mind to provide for the desired bulk,consistency, etc., when combined with an active and the other componentsof a given composition. Typical pharmaceutical carriers include, but arenot limited to, binding agents (including, but not limited topregelatinized maize starch, polyvinylpyrrolidone or hydroxypropylmethylcellulose); fillers (including but not limited to lactose andother sugars, microcrystalline cellulose, pectin, gelatin, calciumsulfate, ethyl cellulose, polyacrylates or calcium hydrogen phosphate.);lubricants (including, but not limited to magnesium stearate, talc,silica, sollidal silicon dioxide, stearic acid, metallic stearates,hydrogenated vegetable oils, corn starch, polyethylene glycols, sodiumbenzoate, sodium acetate); disintegrants (including but not limited tostarch, sodium starch glycolate) and wetting agents (including but notlimited to sodium lauryl sulfate). Additional suitable carriers for thecompositions of the present invention include, but are not limited to,water, salt solutions, alcohol, vegetable oils, polyethylene glycols,gelatin, lactose, amylose, magnesium stearate, talc, silicic acid,viscous paraffin, perfume oil; fatty acid monoglycerides anddiglycerides, petroethral fatty acid esters, hydroxymethylcellulose,polyvinylpyrrolidone, and the like. The pharmaceutical preparations canbe sterilized and if desired, mixed with auxiliary agents, e.g.,lubricants, preservatives, stabilizers, wetting agents, emulsifiers,salts for influencing osmotic pressure, buffers, colorings, flavoringand/or aromatic substances and the like which do not deleteriously reactwith the active compounds.

According to some embodiments, the pharmaceutically acceptable carrierof the compositions of the present invention includes a release agentsuch as a sustained release or delayed release carrier. According tosuch embodiments, the carrier can be any material capable of sustainedor delayed release of the active ingredient to provide a more efficientadministration, resulting in less frequent and/or decreased dosage ofthe active ingredient, ease of handling, and extended or delayedeffects. Non-limiting examples of such carriers include liposomes,microsponges, microspheres, or microcapsules of natural and syntheticpolymers and the like. Liposomes may be formed from a variety ofphospholipids such as cholesterol, stearylamines orphosphatidylcholines.

Additional compositions of the present invention can be prepared readilyusing known technology, such as that which is described in Remington'sPharmaceutical Sciences, 18th or 19th editions, published by the MackPublishing Company of Easton, Pa., which is incorporated herein byreference.

According to some embodiments, the compositions of the present inventioncan further include one or more compatible active ingredients aimed atproviding the composition with another pharmaceutical effect.“Compatible” as used herein means that the active ingredients of such acomposition are capable of being combined with each other in such amanner so that there is no interaction that would substantially reducethe efficacy of each active ingredient or the composition under ordinaryuse conditions.

According to another embodiment of the present invention, thecomposition may be administered serially or in combination with othercompositions for treating conditions of blood loss or anemia or otherblood disorders.

An amount adequate to accomplish therapeutic or prophylactic treatmentis defined herein as a therapeutically-effective dose. In therapeuticregimes, an amount of the compositions of the described invention isadministered until a sufficient beneficial response has been achieved.For example, the response is monitored and repeated dosages are given ifthe response starts to wane. A skilled artisan can determine apharmaceutically effective amount of the inventive compositions bydetermining the dose in a dosage unit (meaning unit of use) that elicitsa given intensity of effect, hereinafter referred to as the “unit dose.”The term “dose-intensity relationship” refers to the manner in which theintensity of effect in an individual recipient relates to dose. Theintensity of effect generally designated is 50% of maximum intensity.The corresponding dose is called the 50% effective dose or individualED50. The use of the term “individual” distinguishes the ED50 based onthe intensity of effect as used herein from the median effective dose,also abbreviated ED50, determined from frequency of response data in apopulation. “Efficacy” as used herein refers to the property of thecompositions of the present invention to achieve the desired response,and “maximum efficacy” refers to the maximum achievable effect. Theamount of the active complex in the compositions of the describedinvention which will be effective in the treatment of a particulardisorder or condition will depend on the nature of the disorder orcondition, and can be determined by standard clinical techniques. (See,for example, Goodman and Gilman's THE PHARMACOLOGICAL BASIS OFTHERAPEUTICS, Joel G. Harman, Lee E. Limbird, Eds.; McGraw Hill, N. Y.,2001; THE PHYSICIAN'S DESK REFERENCE, Medical Economics Company, Inc.,Oradell, N. J., 1995; and DRUG FACTS AND COMPARISONS, FACTS ANDCOMPARISONS, INC., St. Louis, Mo., 1993). The precise dose to beemployed in the formulation will also depend on the route ofadministration, and the seriousness of the disease or disorder, andshould be decided according to the judgment of the practitioner and eachpatient's circumstances. Various administration patterns will beapparent to those skilled in the art.

The dosage ranges for the administration of the compositions of thepresent invention are those large enough to produce the desiredtherapeutic effect.

Those skilled in the art will recognize that initial indications of theappropriate therapeutic dosage of the compositions of the invention canbe determined in in vitro and in vivo animal model systems, and in humanclinical trials. One of skill in the art would know to use animalstudies and human experience to identify a dosage that can safely beadministered without generating toxicity or other side effects. Foracute treatment, it is preferred that the therapeutic dosage be close tothe maximum tolerated dose. For chronic preventive use, lower dosagesmay be desirable because of concerns about long term effects.

Where a range of values is provided, it is understood that eachintervening value, to the tenth of the unit of the lower limit unlessthe context clearly dictates otherwise, between the upper and lowerlimit of that range and any other stated or intervening value in thatstated range is encompassed within the invention. The upper and lowerlimits of these smaller ranges which may independently be included inthe smaller ranges is also encompassed within the invention, subject toany specifically excluded limit in the stated range. Where the statedrange includes one or both of the limits, ranges excluding either bothof those included limits are also included in the invention.

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs. Although any methods andmaterials similar or equivalent to those described herein can also beused in the practice or testing of the present invention, exemplarymethods and materials have been described. All publications mentionedherein are incorporated herein by reference to disclose and describedthe methods and/or materials in connection with which the publicationsare cited.

It must be noted that as used herein and in the appended claims, thesingular forms “a”, “and”, and “the” include plural references unlessthe context clearly dictates otherwise.

The publications discussed herein are provided solely for theirdisclosure prior to the filing date of the present application and eachis incorporated by reference in its entirety. Nothing herein is to beconstrued as an admission that the present invention is not entitled toantedate such publication by virtue of prior invention. Further, thedates of publication provided may be different from the actualpublication dates which may need to be independently confirmed.

Example

The following examples are put forth so as to provide those of ordinaryskill in the art with a complete disclosure and description of how tomake and use the present invention, and are not intended to limit thescope of what the inventors regard as their invention nor are theyintended to represent that the experiments below are all or the onlyexperiments performed. Efforts have been made to ensure accuracy withrespect to numbers used (e.g. amounts, temperature, etc.) but someexperimental errors and deviations should be accounted for. Unlessindicated otherwise, parts are parts by weight, molecular weight isweight average molecular weight, temperature is in degrees Centigrade,and pressure is at or near atmospheric.

Construction of Recombinant ELP Genes Encoding for ELPs

To generate ELPs of a specific and pre-determined chain length, thefollowing is an example of a plasmid reconstruction recursivedirectional ligation (preRDL) strategy can be employed (McDaniel J R,Biomacromolecules. 2010, 11:944-952). Two cloning vectors, which containan ELP gene are cut with two separate sets of restriction enzymes, as isdescribed previously (Sun G, Journal of controlled release: officialjournal of the Controlled Release Society. 2011; 155:218-226), and twovectors are digested with two sets of restriction enzymes, respectively.The two sets of cut vectors are gel purified and ligated together usingan appropriate DNA ligase, resulting in the recursive extension of thegenes encoding for pentameric repeats, for example, (VPGXaaG)n, whereinXaa denotes any amino acid and n denotes the number of repetitivemotifs. The same strategy is employed to generate the ELP diblockcopolymer, where the N-terminal gene of one monoblock is ligated to aC-terminal ELP gene of another via preRDL. For example, the ELP diblockcopolymer comprises one hydrophobic block, comprising (VPGXaaG)n whereinXaa is one of hydrophobic amino acids, n=40-60; and one hydrophilicblock, comprising (VPGXaaG)n wherein Xaa=one of hydrophilic amino acids,n=40-60. Gene sequences encoding for the desired polypeptides can beconfirmed, for example, using diagnostic DNA digestion and DNAsequencing from both N and C termini.

Exemplary Protocol for the Bacterial Transformation of DNA Containingthe Desired Nucleotide Sequence

During ligation, Top10/BLR cells (e.g. Life Technologies, MerckMillipore) are removed from the −80° C. fridge and thawed on ice. Analiquot of 125 μL of the Top10/BLR cells is transferred into a freshtube and an aliquot of 5 μL of DNA ligase is added. The cells in thetube are well mixed by pipetting up and down, and incubated on ice for5-10 min. The cells in the tube are heat shocked for 1 min at 42° C. (or3 min at 37° C.), and incubated for 5 min on ice. The cells are platedonto pre-warmed ampicillin plates, and incubated (upside-down) overnightin the 37° C. incubator.

Expression of ELP Genes and Purification of Recombinant ELPs

The expression vectors containing the desired constructs are transformedinto E. coli cells for protein hyperexpression and proteins are purifiedby inverse transition cycling (ITC). (Golemis E, Adams P D.Protein-protein interactions: a molecular cloning manual. Edn. 2nd ColdSpring Harbor Laboratory Press; Cold Spring Harbor, N.Y.: 200532).Briefly, overnight cultures are spun down and re-suspended in cold PBS.The proteins are liberated from bacteria by periodic probe-tipsonication for an appropriate time period. Insoluble debris is collectedby centrifugation for at 4° C. for an appropriate time period, and thesupernatant is transferred to another tube. Excess poly-ethylene imine(PEI) (MW=3,000) is added to precipitate nucleic acids and the solutionis centrifuged. The supernatant, containing soluble ELP, is purifiedusing about 4-6 rounds of inverse transition cycling (ITC). For eachround, the supernatant is heated to 37° C. to induce phase separation,and the coacervate is collected by centrifugation. The ELP is thenre-suspended in cold PBS and centrifuged at 4° C. again, completing oneround of ITC. About 4-6 rounds of ITC are sufficient to ensure thepurity.

Exemplary Protocol for the Purification of ELPs

An aliquot of 125 μl of BLR cells is transferred into a fresh tube andthawed on ice. An aliquot of 1 μl of miniprep plasmid is transferredinto the tube, which contains the freshly thawed BLR cells (E. colistrain preferred for protein expression) on ice. The tube containingminiprep plasmid and BLR cells is kept on ice for ˜10 min, then movedonto a heat block to heat at 42° C. for 1 min; and then moved into anice bath and kept for 5 min. The cells with recombinant DNA plasmids arethen plated on Amp plates and are incubated overnight upside down.

One colony is selected from the overnight Amp plate, transferred into anErlenmeyer flask, which contains 50 ml of TB media with 50 μlampicillin, and then left overnight in a shaking incubator at 37° C. and250 rpm. 0.75 ml of the overnight culture is added to 0.75 ml DMSOsolution to make a DMSO stock of the overnight culture. The DMSO stockis stored at −80° C. The rest of the culture is centrifuged at 3000 rpm,4° C. for 10 minutes to sediment the bacteria. The bacteria pellet isre-solubilized with an aliquot of 5 ml of media and well mixed bypipetting up and down. An aliquot of 500 μl of re-suspended pellet isinoculated into a one liter (1 L) culture media in a 4 liter Erlenmeyerflask, to which an aliquot of 1 ml of ampicillin is added. TheErlenmeyer flask is then left overnight in a shaking incubator at 37° C.and 250 rpm.

After overnight incubation, the culture is centrifuged at 3000 rpm and4° C. for 10 min to separate the bacteria containing the ELP from thesupernatant. The supernatant is discarded. The pellet is transferredinto a 50 ml conical tube (Falcon tube), which contains 40 ml of coldPBS to re-suspend and form a suspension, which is then vortexed. Theconical flask is then put in a plastic beaker, which contains ice and alittle bit of water and is sonicated according to the following timesequence to generate a lysed product, (i.e., 10 secs on, 20 secs off,repeat cycle for 3 mins).

The lysed product is transferred from the Falcon tube to an Oakridgetube and then is cold spin sonicated at 12,000 rpm, 4° C., for 15 min.The supernatant is transferred to a 50 ml Falcon tube and the pellet,which contains insoluble cellular debris, is discarded. PEI is added tothe supernatant to a final concentration of 0.5% and mixed gently. Thesolution is then incubated for 10-20 min on ice, with occasional gentlemixing, and then centrifuged at 12,000 rpm for 15 min at 4° C. Thesupernatant is transferred to a new centrifuge tube (Falcon tube); thepellet, which contains precipitated DNA and insoluble cellular debris,is discarded. The supernatant is placed in a 37° C. water bath for 10min, and then, about 2.6 g of NaCl (1M of NaCl in ˜45 ml) is added tothe supernatant. The supernatant is then spun at 37° C., 4000 rpm for 10min. The supernatant is removed and the pellet is re-suspended on ice in15 ml of cold PBS; the supernatant is then transferred to the Oakridgetube and re-suspended in 5 ml of PBS first, and then washed out with 10ml of PBS. The suspension is then spun at 12,000 rpm, 4° C., for 10 minto remove any remaining insoluble matter. The supernatant is retainedand any pellet formed is discarded.

One exemplary ELP diblock copolymer comprises one hydrophobic block,comprising (VPGXaaG)n wherein Xaa is Ile and n=48; and one hydrophilicblock, comprising (VPGXaaG)n wherein Xaa=Ser and n=48. The amino acidsequence of the exemplary ELP diblock copolymer is shown as SEQ ID NO.7.

ELP Spherical Nanoparticles Possessing Hemoglobin, a Subunit(s), aFragment(s), a Derivative(s), or a Functional Equivalent Thereof at theCore of ELP Via Noncovalent Attractions

Hemoglobin, a subunit(s), a fragment(s), a derivative(s), or afunctional equivalent thereof is mixed with the recombinant ELP based ona 1:1 molar ratio at a 7.4±0.3 pH in PBS buffer to form anELP-hemoglobin complex, where the ELP and the hemoglobin, subunit(s),fragment(s), derivative(s), or functional equivalent thereof are heldtogether by noncovalent attractions, for example, salt bridge, hydrogenbonds, and/or a hydrophobic effect.

The amino acid sequences of exemplary hemoglobin subunit(s) are SEQ IDNO. 4, SEQ ID NO. 5 and SEQ ID NO. 6. The amino acid sequence of anexemplary ELP is SEQ ID NO. 7.

Fusion Protein Formed by Chemical Reaction of ELP with Hemoglobin, aSubunit(s), a Fragment(s), a Derivative(s), or a Functional EquivalentThereof

Hemoglobin, a subunit(s), a fragment(s), a derivative(s), or afunctional equivalent thereof is mixed with the recombinant ELP based on1:1 molar ratio at a 7.4±0.3 pH in PBS buffer. The reaction is kept at4° C. overnight. Size-exclusion chromatography is used to removeunreacted reagents from the fusion protein consisting of ELP operativelylinked to the hemoglobin, subunit(s), fragment(s), derivative(s), orfunctional equivalent thereof.

The amino acid sequences of exemplary hemoglobin subunit(s) are SEQ IDNO. 4, SEQ ID NO. 5, and SEQ ID NO. 6. The amino acid sequence ofexemplary ELP is SEQ ID NO. 7.

Construction of Fusion Genes Encoding for Fusion Proteins

The gene encoding human hemoglobin, subunit(s), fragment(s),derivative(s), or functional equivalent thereof is inserted into acloning vector, which is linearized. The nucleotide sequence of theexemplary hemoglobin subunit(s) is selected from the group consisting ofSEQ ID. NO. 1, SEQ ID. NO. 2, and SEQ ID. NO. 3. A cassette for the ELPgene encoding ELP diblock copolymer (VPGXaaG)n comprising a hydrophobicblock, wherein Xaa is one of hydrophobic amino acids; and a hydrophilicblock, wherein Xaa is one of hydrophilic amino acids, is removed fromthe cloning vector by double digestion, followed by electrophoreticseparation and agarose gel extraction. The ELP cassette is then ligatedinto the linearized vector operatively linked to a nucleotide sequenceencoding a hemoglobin subunit. The fusion gene cassette is then removedby double digestion, followed by electrophoretic separation and agarosegel extraction. Separately, an expression vector is double digested,treated, agarose gel purified, and then ligated with the fusion genecassette to yield the target fusion gene (ELP-hemoglobin) in anexpression vector. The expression vector containing the target fusiongene (ELP-hemoglobin) is transformed into an expression strain of E.coli.

Expression of Fusion Genes and Purification of Fusion Proteins

An appropriate amount of media with appropriate concentration ofantibiotics, for example, ampicillin is inoculated with the expressionstrain and grown using a hyper expression protocol. (Daniell H, et al.,Methods Mol Biol 1997, 63:359-371). Overnight cell cultures are spundown and re-suspended in cold PBS. Cells are harvested bycentrifugation, re-suspended in cold PBS, lysed by probe-tip sonicationat 4° C., and centrifuged at 4° C. to eliminate insoluble cell debris.The supernatant containing soluble fusion protein is transferred toanother tube, nucleic acids are precipitated using polyethyleneimine(PEI) and removed by centrifugation at 4° C.

Fusion protein (containing ELP-hemoglobin) is purified by 4-6 rounds ofinverse transition cycling (ITC) as described previously (McPherson D T,et al., Protein Expr Purif 1996, 7(1):51-57). Briefly, for one round ofITC, the supernatant, containing the soluble fusion protein is heated toinduce phase separation, and the coacervate is collected bycentrifugation. The fusion protein is then re-suspended in cold PBS andre-centrifuged at 4° C.

To confirm fusion protein purity, for example, SDS-PAGE can beperformed. To determine concentrations of fusion protein, for example,the concentration can be determined spectrophotometrically usingcalculated extinction coefficients (Gill S, et al., AnalyticalBiochemistry 1989, 182:319-326).

The amino acid sequences of the exemplary fusion proteins are presentedas SEQ ID NO. 8, SEQ ID NO. 9, and SEQ ID NO. 10. For example, thefusion protein is of amino acid sequence SEQ ID No. 8 comprising an ELPamino acid sequence SEQ ID No. 7 operatively linked to a hemoglobinamino acid sequence SEQ ID No. 4. The fusion protein of amino acidsequence SEQ ID No. 9 comprises an ELP amino acid sequence SEQ ID No. 7operatively linked to a hemoglobin amino acid sequence SEQ ID No. 5. Thefusion protein of amino acid sequence SEQ ID No. 8 comprises an ELPamino acid sequence SEQ ID No. 7 operatively linked to a hemoglobinamino acid sequence SEQ ID No. 6. According to some embodiments, alinking peptide (Yaa)m resides between the ELP amino acid sequence andhemoglobin amino sequence, wherein Yaa specifies any amino acid and mdenotes a number of repetitive amino acids.

Isolation of Plasmid DNA Containing ELP-Hemoglobin Fusion Gene

Plasmid DNA containing an ELP-hemoglobin fusion gene can be isolated,for example, using a Qiagen minprep kit according to manufacturer'sprotocol. Briefly, plastic culture tubes are filled with 4 mL ofautoclaved TB media and inoculated with a bacterial colony transformedwith plasmid DNA containing an ELP-hemoglobin fusion gene. Next, theinoculated tubes are incubated overnight at 37° C. in a shakerincubator. The next day, four, 1.5 mL tubes and 1 filter are labeled foreach colony selected. After incubating overnight, 0.5 mL aliquots areremoved from the inoculated tubes and are transferred to the newlylabeled 1.5 mL tubes. The inoculated tubes are centrifuged for 10minutes at 4,000 rpm. After centrifugation, supernatant is discarded,the pellets are resuspended in 250 μL Buffer P1 (Qiagen, Valencia,Calif.), and the resuspended pellets are transferred to a 1.5 mLmicrofuge tube. Next, 250 μL Buffer P2 (Qiagen, Valencia, Calif.) isadded to the microfuge tube containing Buffer P1 and the tube isinverted 4-6 times. After inversion, 350 μL Buffer N3 (Qiagen, Valencia,Calif.) is added to the microfuge tube containing Buffer P1 and BufferP2 and the tube is inverted 4-6 times. Following inversion, themicrofuge tube is centrifuged in a table-top centrifuge for 10 minutesat 13.2 rpm. After centrifugation, the supernatant is poured into theappropriate pre-labeled filter and centrifuged in a table-top centrifugefor 0.5 minutes at 13.2 rpm. After centrifugation, the flow-through isdiscarded and 750 μL Buffer PE (Qiagen, Valencia, Calif.) is added tothe filter and the filter is centrifuged in a table-top centrifuge or0.5 minutes at 13.2 rpm. The flow-through is dicared and the filter iscentrifuged in a table-top centrifuge or 0.5 minutes at 13.2 rpm. Aftercentrifugation, the top half of filter is placed in the appropriatepre-labeled microfuge tube; the bottom half of the filter is discarded.Next, 50 μL of autoclaved water is added to the filter and the filter isincubated at room temperature for 2-3 minutes before centrifuging in atable-top centrifuge or 0.5 minutes at 13.2 rpm. After centrifugation,the flow-through is collected and the filter is discarded. An opticaldensity (OD) measurement at 280 nm can be performed on the flow-throughto determine plasmid DNA concentration.

Expression of ELP-Hemoglobin Fusion Protein

ELP-hemoglobin fusion protein can be expressed, for example, by E. coliBLR cells using the following exemplary protocol. Briefly, 125 μL offreshly thawed BLR cells are transformed with 1 μL of isolated plasmidcontaining an ELP-hemoglobin fusion gene by incubating on ice for 10minutes, in a 42° C. heat block for 1 minute and on ice for 5 minutes.Following transformation, the BLR cells are plated on an ampicillin(Amp) agar plate and incubated upsided down overnight. Followingovernight incubation, one colony is selected from the Amp agar plate andis placed in Erlenmyer flask containing 50 mL of TB media and 50 μL ofampicillin. The flask is incubated overnight in a shaker incubator at37° C. and 250 rpm. After overnight incubation, a DMSO stock of theculture is prepared by adding 0.75 mL of culture to 0.75 mL DMSO. TheDMSO stock is then placed in a −80° C. freezer. The remaining overnightculture is centrifuged at 3,000 rpm at 4° C. for 10 minutes. Supernatantis discarded and the pellet is resuspended in 5 mL TB media. Next, 500μL of the resuspended culture is used to inoculate a 4 L Erlenmeyerflask containing 1 L of TB media and 1 mL ampicillin. The inoculatedflask is incubated overnight in a shaker incubator at 37° C. and 250rpm. Next, the overnight culture is centrifuged at 3,000 rpm, at 4° C.for 10 minutes. The supernatant is discarded, the cell pellet isresuspend with 40 mL cold PBS, vortexed and sonicated to lyse the cells.The lysed cells are placed in an ice bath for 10 seconds, removed fromthe ice for 20 seconds and this cycle is repeated for a total of 3minutes. The lysed cells are transferred to an Oakridge tube andcentrifuged at 12,000 rpm at 4° C. for 15 minutes. After centrifugation,the pellet is discarded and the supernatant is transferred to a 50 mLconical tube. Polyethyleneimine (PEI) is added to the supernatant at afinal concentration of 0.5% and gently mixed. The supernatant is thenincubated on ice for 10-20 minutes, with occasional gentle mixing. Afterincubation, the supernatant is centrifuged at 12,000 rpm for 15 minutesat 4° C. After centrifugation, the pellet is discarded and thesupernatant is transferred to new 50 mL conical tube. The supernatant isplaced in a 37° C. water bath, 2.6 g of NaCl is added, and thesupernatant is incubated for 10 minutes. Following incubation, thesupernatant is centrifuged at 4,000 rpm for 10 minutes at 37° C. Thesupernatant is discarded and the pellet is resuspended on ice in 15 mLof cold PBS. The resuspended pellet is transferred to an Oakridge tubeand centrifuged at 12,000 rpm for 10 minutes at 4° C. The pellet isdiscarded and the supernatant is retained. Again, the supernatant isplaced in a 37° C. water bath, 2.6 g of NaCl is added, and thesupernatant is incubated for 10 minutes. Following incubation, thesupernatant is centrifuged at 4,000 rpm for 10 minutes at 37° C. Thesupernatant is discarded and the pellet is resuspended on ice in 15 mLof cold PBS. The resuspended pellet is transferred to an Oakridge tubeand centrifuged at 12,000 rpm for 10 minutes at 4° C. The pellet isdiscarded and the supernatant is retained.

Chemical Conjugation of Elastin-Like Protein (ELP) to Hemoglobin

ELP was conjugated to hemoglobin using a1-Ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC) based linkeraccording to the manufacturer's (Thermo Scientific, Grand Island, N.Y.)protocol.

Briefly, a 10-fold molar excess of EDC (Thermo Scientific, ProductNumber 22980) was added directly to hemoglobin (Sigma-Aldrich, CatalogNumber H7379). Next, 0.6 mg of N-hydroxysuccinimide (NHS) was added tothe EDC and hemoglobin, the components were mixed and allowed to reactfor 15 minutes at room temperature. After the 15 minute reaction, ELPexpressed in E. coli (as described above) was added to the hemoglobinreaction mixture to produce hemoglobin:ELP ratios of 2:1, 1;1 and 1:4.Next, the ELP and hemoglobin reaction mixture was incubated for 2 hoursat room temperature. After the 2 hour incubation, the reaction wasquenched by adding hydroxylamine to a final concentration of 10 mM.

SDS-PAGE of ELP-Hemoglobin Fusion Proteins

ELP-hemoglobin fusion was assessed by sodium dodecyl sulfatepolyacrylamide gel electrophoresis (SDS-PAGE).

Briefly, ELP-hemoglobin samples containing hemoglobin:ELP ratios of 2:1,1:1 and 1:4 were mixed with 1× Sample Buffer (62.5 mM Tris HCl, pH 6.8at 25° C.; 2% w/v SDS; 10% Glycerol; 50 mM DTT; 0.01% w/v BromophenolBlue). Approximately 20 ug of each ELP-hemoglobin protein was resolved(125V, 30-40 mA) on a SDS-PAGE gel (Thermo Scientific, Grand Island,N.Y.). Next, the gel was washed 2×5 minutes in ultrapure water, fixed2×15 minutes in 30% ethanol:10% acetic acid solution, and washed 2×5minutes in 10% ethanol then 2×5 minutes in ultrapure water. After thegel was fixed and washed, the gel was sensitized for 1 minute in SilverStain Sensitizer (Thermo Scientific, Product Number 24612) and thenwashed 2×1 minute with water. Next, the gel was stained in Silver StainWorking Solution (0.5 mL enhancer with 25 mL Stain) (Thermo Scientific,Product Number 24612) for 30 minutes. After the gel was stained, the gelwas washed 2×20 seconds with ultrapure water and developed for 2-3minutes (or until bands appear) using Developer Working Solution (0.5 mLEnhancer with 25 mL Developer) (Thermo Scientific, Product Number24612). Finally, the developing reaction was stop by adding 5% aceticacid for 10 minutes.

FIG. 6 shows the results SDS-PAGE. Without being bound by theory, thetwo protein bands directly above the ELP bands in lanes 1-6 are believeto correspond to ELP binding either one subunit or two subunits ofhemoglobin.

Size Exclusion Analysis of ELP-Hemoglobin

Size exclusion analysis was performed on ELP-hemoglobin obtained by thechemical conjugation method described above.

Briefly, ELP-hemoglobin (1:4 ratio of hemoglobin:ELP) wasinjected/loaded onto a size exclusion column using a BioLogic DuoFlowChromatography System (Bio-Rad, Hercules, Calif.). The load/injectionparameters were as follows:

Static Loop

Buffer A: 100% (1×PBS)/Buffer B: 0% (ddH₂0)

20 mL (4× of sample loop volume)/2.60 mL/min.

Once the ELP-hemoglobin sample was loaded on the column, the sample wasrun through the column using the following parameters:

Isocratic Flow; Buffer A: 100%/Buffer B: 0%; 480 mL/2.60 mL/min.

Isocratic Flow; Buffer A: 0%/Buffer B: 100%; 850 mL/2.60 mL/min.

Isocratic Flow; buffer A: 100%/Buffer B: 0%; 350 mL/2.60 mL/min.

Once a peak was observed, samples were collected using a fractioncollector.

FIG. 7 shows the chromatogram of the size exclusion analysis performedon the ELP-hemoglobin fusion protein (1:4 ratio of hemoglobin:ELP). Thefirst peak (Fraction 1) is ELP-hemoglobin fusion. The second peak(Fraction 2) is ELP.

Dynamic Light Scattering (DLS) Analysis

A dynamic light scattering (DLS) instrument (Wyatt Technology, SantaBarbara, Calif.) was used to measure the hydrodynamic radium of the twofractions (Fraction 1 and Fraction 2) collected by size exclusionanalysis.

FIG. 8 shows a bar graph (intensity (%) vs. radium (nm)) of dynamiclight scattering (DLS) results for Fraction 1 (first peak) and Fraction2 (second peak) collected by size exclusion analysis. The hydrodynamicradius of Fraction 1 was equal to 11.4 nm. The hydrodynamic radius ofFraction 2 was equal to 7.4 nm.

FIG. 9 shows a line graph (intensity (%) vs. radium (nm)) of the dynamiclight scattering (DLS) results for the ELP-hemoglobin fusion protein. Anincrease in hydrodynamic radius of the ELP-hemoglobin fusion is observedwhen compared to hemoglobin and to ELP.

UV-Vis Characterization

UV-vis was used to determine whether absorption at 400 nm by hemoglobinwas maintained in Fraction 1 (first peak/ELP-hemoglobin) collected bysize exclusion analysis.

Briefly, both the visible lamp and the UV lamp of a scanningspectrophotometer (Beckman Coulter, Fullerton, Calif.) were switched onto warm. Next, the analysis method was set to “Wavelength Scan II”. Acuvette containing blank solvent was placed in the first position of thecuvette holder. Cuvettes containing hemoglobin, Fraction 1 (firstpeak/ELP-hemoglobin) and Fraction 2 (second peak/ELP) were placed inpositions 2, 3 and 4 respectively. The instrument was blanked using theblank solvent cuvette and then the cuvettes containing hemoglobin,Fraction 1 and Fraction 2 were scanned over a series of wavelengthsranging from 200 nm to 800 nm.

FIG. 10 shows the UV-vis results (absorbance vs. wavelength in nm) forhemoglobin, Fraction 1 (first peak) and Fraction 2 (second peak). Theseresults indicate that 400 nm absorption of hemoglobin was maintainedafter ELP modification.

Phase Separation of ELP

An important property of ELP is its ability to undergo phase separation.That is, below a critical transition temperature, ELP is a solubleunimer in aqueous solution, whereas above its transition temperature,ELP undergoes a phase transition and aggregates into an insolublecoacervate (Urry D W, J. Phys. Chem. B. 1997; 101: 11007-11028). Becausethe fusion of ELP to a protein can alter the phase behavior of ELP,phase separation of Fraction 1 (first peak/ELP-hemoglobin) and Fraction2 (second peak/ELP) was measured by temperature-programmed turbidimetry.Briefly, light attenuation of Fraction 1 and Fraction 2 collected bysize exclusion analysis was monitored at 350 nm as the temperature wasramped at a rate of 1° C./min.

FIG. 11 shows the phase separation results (absorbance at 350 nm vs.Temperature in ° C.) for Fraction 1 (first peak/ELP-hemoglobin) andFraction 2 (second peak/ELP) collected by size exclusion analysis. Thephase separation results indicate that ELP phase separation ismaintained after ELP-hemoglobin fusion.

While the present invention has been described with reference to thespecific embodiments thereof it should be understood by those skilled inthe art that various changes may be made and equivalents may besubstituted without departing from the true spirit and scope of theinvention. In addition, many modifications may be made to adopt aparticular situation, material, composition of matter, process, processstep or steps, to the objective spirit and scope of the presentinvention. All such modifications are intended to be within the scope ofthe claims appended hereto.

What is claimed is:
 1. A biocompatible pharmaceutical compositioncomprising a therapeutic amount of a complex comprising a polymer inassociation with a hemoglobin (Hb), a Hb subunit(s), a Hb fragment(s), aHb derivative(s), or a functional equivalent thereof that stores andreleases oxygen in accordance with an oxygen dissociation curve; whereinthe therapeutic amount of the complex is effective to treat a conditioncaused by blood loss, anemia, or a hemoglobin disorder, and to improvesubject survival relative to a control, wherein the polymer is a proteinpolymer, a polynucleotide polymer, a polysaccharide polymer, or asynthetic polymer.
 2. The biocompatible pharmaceutical composition ofclaim 1, wherein the condition caused by blood loss includes hemorrhagicshock.
 3. The biocompatible pharmaceutical composition of claim 2, theprotein polymer is associated with the Hb, the Hb subunit(s), the Hbfragment(s), the Hb derivative(s), or the functional equivalent thereofvia a covalent bond, an ionic bond, a hydrogen bond, a hydrophobicforce, encapsulation, or via fusion.
 4. The biocompatible pharmaceuticalcomposition of claim 1, wherein the protein polymer is an elastin-likepolypeptide (ELP).
 5. The biocompatible pharmaceutical composition ofclaim 4, wherein the ELP and the Hb, the Hb subunit(s), the Hbfragment(s), the Hb derivative(s), or the functional equivalent thereofare operatively linked to form a fusion protein, which is encoded by apolynucleotide comprising a nucleotide sequence that encodes the ELP anda nucleotide sequence that encodes the Hb, the Hb subunit(s), the Hbfragment(s), the Hb derivative(s), or the functional equivalent thereof.6. The biocompatible pharmaceutical composition of claim 4, wherein theELP and the Hb, the Hb subunit(s), the Hb fragment(s), the Hbderivative(s), or the functional equivalent thereof are operativelylinked to form a fusion protein, which is obtained by chemically joiningthe ELP and the ELP and the Hb, the Hb subunit(s), the Hb fragment(s),the Hb derivative(s), or the functional equivalent thereof.
 7. Thebiocompatible pharmaceutical composition of claim 4, wherein the ELP andthe Hb, the Hb subunit(s), the Hb fragment(s), the Hb derivative(s), orthe functional equivalent thereof are operatively linked to form acomplex, wherein the ELP is assembled into a spherical nanoparticlecomprising a core into which the Hb, the Hb subunit(s), the Hbfragment(s), the Hb derivative(s), or the functional equivalent thereofis encapsulated.
 8. The biocompatible pharmaceutical composition ofclaim 5, wherein the fusion protein is assembled into a sphericalnanoparticle comprising a core inside of which the Hb, the Hbsubunit(s), the Hb fragment(s), the Hb derivative(s), or the functionalequivalent thereof is enclosed.
 9. The biocompatible pharmaceuticalcomposition of claim 4, wherein the ELP comprises a pentameric aminoacid motif (Val-Pro-Gly-Xaa-Gly)_(n), wherein Xaa specifies any aminoacid and n denotes a number of repetitive motifs.
 10. The biocompatiblepharmaceutical composition of claim 9, wherein n=20-90, and Xaa isSerine or a conservative amino acid substitute thereof.
 11. Thebiocompatible pharmaceutical composition of claim 10, wherein theconservative amino acid substitute of Serine is Thr.
 12. Thebiocompatible pharmaceutical composition of claim 9, wherein n=20-90,and Xaa is Isoleucine or a conservative amino acid substitute thereof.13. The biocompatible pharmaceutical composition of claim 11, whereinthe conservative amino acid substitute of Isoleucine is Leu or Met orVal.
 14. The biocompatible pharmaceutical composition of claim 4,wherein the ELP comprises a diblock copolymer comprising: a hydrophilicblock comprising a pentameric amino acid motif(Val-Pro-Gly-Xaa-Gly)_(n), wherein n=20-90, and Xaa is a hydrophilicamino acid; and a hydrophobic block comprising a pentameric amino acidmotif (Val-Pro-Gly-Xaa-Gly)_(n), wherein n=20-90, and Xaa is ahydrophobic amino acid.
 15. The biocompatible pharmaceutical compositionof claim 14, wherein for the hydrophilic block, the Xaa is selected fromthe group consisting of lysine (+), arginine (+), aspartate (−) andglutamate (−), serine, threonine, asparagine, glutamine, and histidine;and for the hydrophobic block, Xaa is selected from the group consistingof alanine, valine, leucine, isoleucine, proline, phenylalanine,tryptophan, and methionine.
 16. The biocompatible pharmaceuticalcomposition of claim 15, wherein for the hydrophilic block the Xaa isSerine or a conservative amino acid substitute thereof; and for thehydrophobic block the Xaa is Isoleucine or a conservative amino acidsubstitute thereof.
 17. The biocompatible pharmaceutical composition ofclaim 16, wherein the conservative amino acid substitute of Serine isThr; and the conservative amino acid substitute of Isoleucine is Leu orMet or Val.
 18. The biocompatible pharmaceutical composition of claim14, wherein n=48 for hydrophobic block and n=48 for hydrophilic block.19. The biocompatible pharmaceutical composition of claim 14, whereinthe Hb, the Hb subunit(s), the Hb fragment(s), the Hb derivative(s), orthe functional equivalent thereof is operatively linked to theC-terminus of the ELP.
 20. The biocompatible pharmaceutical compositionof claim 14, wherein the Hb, the Hb subunit(s), the Hb fragment(s), theHb derivative(s), or the functional equivalent thereof is operativelylinked to the hydrophobic block of the ELP.
 21. The biocompatiblepharmaceutical composition of claim 1, wherein the Hb, the Hbsubunit(s), the Hb fragment(s), the Hb derivative(s), or the functionalequivalent thereof is of an amino acid sequence selected from the groupconsisting of SEQ ID No. 4, SEQ ID No. 5 and SEQ ID No.
 6. 22. Thebiocompatible pharmaceutical composition of claim 14, wherein the ELP isof amino acid sequence SEQ ID NO.
 7. 23. The biocompatiblepharmaceutical composition of claim 1, wherein the Hb, the Hbsubunit(s), the Hb fragment(s), the Hb derivative(s), or the functionalequivalent thereof is encoded by a polynucleotide sequence selected fromthe group consisting of SEQ ID No. 1, SEQ ID No. 2 and SEQ ID No.
 3. 24.The biocompatible pharmaceutical composition of claim 1, furthercomprising one or more pharmaceutically acceptable salts.
 25. A methodof treating a condition due to blood loss and improving subjectsurvival, the method comprising: (1) administering a biocompatiblepharmaceutical composition comprising a therapeutic amount of a complexcomprising a polymer associated with a Hb, subunit(s), a Hb fragment(s),a Hb derivative(s), or a functional equivalent thereof, wherein thepolymer is a protein polymer, a polynucleotide polymer, a polysaccharidepolymer, or a synthetic polymer; wherein the therapeutic amount iseffective to store and release oxygen in accordance with an oxygendissociation curve.
 26. The method of claim 25, wherein the conditioncaused by blood loss includes hemorrhagic shock.
 27. The method of claim26, wherein the protein polymer is associated with the Hb, the Hbsubunit(s), the Hb fragment(s), the Hb derivative(s), or the functionalequivalent thereof via a covalent bond, an ionic bond, a hydrogen bond,a hydrophobic force, encapsulation, or via fusion.
 28. The method ofclaim 25, wherein the protein polymer is an elastin-like polypeptide(ELP).
 29. The method of claim 28, wherein the ELP and the Hb, the Hbsubunit(s), the Hb fragment(s), the Hb derivative(s), or the functionalequivalent thereof are operatively linked to form a fusion protein,which is encoded by a polynucleotide comprising a nucleotide sequencethat encodes the ELP and a nucleotide sequence that encodes the Hb, theHb subunit(s), the Hb fragment(s), the Hb derivative(s), or thefunctional equivalent thereof.
 30. The method of claim 28, wherein theELP and the Hb, the Hb subunit(s), the Hb fragment(s), the Hbderivative(s), or the functional equivalent thereof are operativelylinked to form a fusion protein, which is obtained by chemically joiningthe ELP and the ELP and the Hb, the Hb subunit(s), the Hb fragment(s),the Hb derivative(s), or the functional equivalent thereof.
 31. Themethod of claim 28, wherein the ELP is assembled into a sphericalnanoparticle comprising a core inside of which the Hb, the Hbsubunit(s), the Hb fragment(s), the Hb derivative(s), or the functionalequivalent thereof is encapsulated.
 32. The method of claim 28, whereinthe fusion protein is assembled into a spherical nanoparticle comprisinga core inside of which the Hb, the Hb subunit(s), the Hb fragment(s),the Hb derivative(s), or the functional equivalent thereof is enclosed.33. The method of claim 28, wherein the ELP comprises a pentameric aminoacid motif (Val-Pro-Gly-Xaa-Gly)_(n), wherein Xaa specifies any aminoacid and n denotes a number of repetitive motifs.
 34. The method ofclaim 33, wherein n=20-90, and Xaa is Serine or a conservative aminoacid substitute thereof.
 35. The method of claim 34, wherein theconservative amino acid substitute of Serine is Thr.
 36. The method ofclaim 33, wherein n=20-90, and Xaa is Isoleucine or a conservative aminoacid substitute thereof.
 37. The method of claim 36, wherein theconservative amino acid substitute of Isoleucine is Leu or Met or Val.38. The method of claim 28, wherein the ELP comprises a diblockcopolymer comprising: a hydrophilic block comprising a pentameric aminoacid motif (Val-Pro-Gly-Xaa-Gly)_(n), wherein n=20-80, and Xaa is ahydrophilic amino acid; and a hydrophobic block comprising a pentamericamino acid motif (Val-Pro-Gly-Xaa-Gly)_(n), wherein n=20-80, and Xaa isa hydrophobic amino acid.
 39. The method of claim 38, wherein the Xaa isselected from the group consisting of lysine (+), arginine (+),aspartate (−) and glutamate (−), serine, threonine, asparagine,glutamine, and histidine in the hydrophilic block; and Xaa is selectedfrom the group consisting of alanine, valine, leucine, isoleucine,proline, phenylalanine, tryptophan, and methionine in the hydrophobicblock.
 40. The method of claim 39, wherein for the hydrophilic block theXaa is Serine or a conservative amino acid substitute thereof; and forthe hydrophobic block the Xaa is Isoleucine or a conservative amino acidsubstitute thereof.
 41. The method of claim 40, wherein the conservativeamino acid substitute of Serine is Thr, and the conservative amino acidsubstitute of Isoleucine is Leu or Met or Val.
 42. The method of claim38, wherein n=48 for hydrophobic block and n=48 for hydrophilic block.43. The method of claim 38, wherein the Hb, the Hb subunit(s), the Hbfragment(s), the Hb derivative(s), or the functional equivalent thereofis operatively linked to the C-terminus of the ELP.
 44. The method ofclaim 38, wherein the Hb, the Hb subunit(s), the Hb fragment(s), the Hbderivative(s), or the functional equivalent thereof is operativelylinked to the hydrophobic block of the ELP.
 45. The method of claim 25,wherein the Hb, the Hb subunit(s), the Hb fragment(s), the Hbderivative(s), or the functional equivalent thereof is of amino acidsequence selected from the group consisting of SEQ ID No. 4, SEQ ID No.5 and SEQ ID No.
 6. 46. The method of claim 38, wherein the ELP is ofamino acid sequence SEQ ID NO.
 7. 47. The method of claim 25, whereinthe Hb, the Hb subunit(s), the Hb fragment(s), the Hb derivative(s), orthe functional equivalent thereof is encoded by a polynucleotidesequence selected from the group consisting of SEQ ID No. 1, SEQ ID No.2 and SEQ ID No.
 3. 48. The method of claim 25, wherein thebiocompatible pharmaceutical composition further comprises one or morepharmaceutically acceptable salts.
 49. The method of claim 25, furthercomprising constructing a vector and/or host cell comprising a fusiongene polynucleotide that comprises a polynucleotide sequence coding afusion protein comprising ELP and Hb, the Hb subunit(s), the Hbfragment(s), the Hb derivative(s), or the functional equivalent thereof.50. The method of claim 49, further comprising preparing the fusionprotein by expressing the fusion gene polynucleotide in an expressionsystem.
 51. The method of claim 50, further comprising separating orpurifying the fusion protein from the expression system.
 52. The methodof claim 51, further comprising preparing the fusion protein bychemically operatively linking the ELP and Hb, the Hb subunit(s), the Hbfragment(s), the Hb derivative(s), or the functional equivalent thereof.